WO2018074770A1 - Use of rnf20 for diagnosis and treatment of kidney or liver cancer and screening for therapeutic agents for kidney or liver cancer - Google Patents

Use of rnf20 for diagnosis and treatment of kidney or liver cancer and screening for therapeutic agents for kidney or liver cancer Download PDF

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WO2018074770A1
WO2018074770A1 PCT/KR2017/011172 KR2017011172W WO2018074770A1 WO 2018074770 A1 WO2018074770 A1 WO 2018074770A1 KR 2017011172 W KR2017011172 W KR 2017011172W WO 2018074770 A1 WO2018074770 A1 WO 2018074770A1
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rnf20
expression
gene
protein
srebp1c
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김재범
이재호
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서울대학교산학협력단
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Definitions

  • the present application is a technique related to the diagnosis, treatment and treatment drug development of cancer associated with abnormalities of RNF20.
  • Kidney cancer occurs mainly in elderly people in their 60's and 70's, and continues to increase, and Korea ranks 10th with 2.0% of cancers occurring in men and 15th with 1.2% in women.
  • Renal cell carcinoma includes clear cell RCC, papillary RCC, chromophobe RCC, medullary RCC, and nonclassifiable renal cell carcinoma. (unclassified RCC), kidney transitional cell carcinoma (TCC), and renal eosinophilic granuloma (ranal oncocytoma). Clear cell type renal cell carcinoma accounted for 66-75%, papillary renal cell carcinoma accounted for 15%, and anachromosomal renal cell carcinoma accounted for 5%.
  • Kidney cancer has little symptoms when the tumor is small, and symptoms do not appear until the tumor is large enough to push the organs. Therefore, since the diagnosis is often delayed, about 30% of the patients are already metastasized when the first diagnosis is made. Therefore, a method for early diagnosis of kidney cancer is needed.
  • kidney cancer Currently, methods used for diagnosing kidney cancer include abdominal ultrasonography, abdominal computed tomography (CT), etc., and use of expensive equipment has a disadvantage in that it is inexpensive to use for screening purposes.
  • CT computed tomography
  • Hepatocellular carcinoma is also the most common type of adult liver cancer and is the third leading cause of death from cancer (Stefaniuk P, et al., 2010, World J Gastroenterol 16: 418-424). HCC is a disease that manifests only when it progresses considerably, which often leads to a missed time for proper treatment and a very poor prognosis. In particular, surgical removal of the disease is a serious disease that dies within one year. When the diagnostic method is improved, a significant improvement in treatment is expected.
  • Ring finger protein 20 is an E3 ubiquitin ligase that plays a variety of roles in transcriptional regulation, DNA damage response, stem cell differentiation and adipose synthesis.
  • RNF20 promotes monoubiquitination of histone H2B, which regulates transcription of gene subsets and contributes to chromatin remodeling (Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10 , 483-488; Shema et al., 2011).
  • US Patent Publication No. 2011-0081362 relates to the treatment of cancer, a technique for inhibiting modulators involved in a number of intracellular processes including RNF20 to selectively inhibit the growth or survival of cancer cells comprising RAS active mutations. Is disclosed.
  • US Patent Publication No. 2013-0295584 relates to the use of histone protein polyubiquitonation in cancer biomarkers, which uses monopolyquitinization of histone 2B in the identification of thyroid cancer, based on the interaction between CDC73 and RNF20. Disclosed are methods for screening substances that modulate monoubiquitination of histone proteins.
  • the present application seeks to provide cancer therapeutic agents, diagnostic markers and therapeutic screening methods associated with abnormalities of RNF20 based on novel molecular mechanisms.
  • the present application relates to cancer associated with a reduction or deletion of RNF20, including a ring finger protein 20 (RNF20) gene, or a protein encoded by said gene, or a substance that increases expression of said gene, in particular solid cancer, in particular kidney cancer Or it provides a pharmaceutical composition for the prevention or treatment of liver cancer.
  • RNF20 ring finger protein 20
  • the expression of RNF20 is markedly reduced in renal and liver cancers, and tumors are suppressed upon overexpression through inhibition of demethylation or hypermethylation of the gene or protein in vivo and in vitro, or by demethylation of the endogenous RNF20 gene.
  • kidney cancer in which the composition according to the invention is used comprises kidney cancer, in particular not caused by VHL gene mutations.
  • the present invention also provides a composition for the diagnosis or prognosis of cancer, in particular solid cancer, in particular renal cancer or liver cancer, associated with a reduction or deletion of RNF20, comprising a substance for detecting ring finger protein 20 (RNF20).
  • RNF20 can be measured at the gene or protein level, and can be used to detect, judge, diagnose, or predict survival prognosis for kidney or liver cancer.
  • the present invention also relates to a method for screening or preventing or treating cancer, in particular solid cancer, more particularly renal or liver cancer, associated with a reduction or deletion of RNF20 targeting the RNF20-SREBP1c-PTTG1 signaling pathway.
  • the method comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase expression of RNF20; A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And comparing the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance and the control cells not in contact with the test substance, so that the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance is less than that of the control. And a fourth step of selecting it as a therapeutic agent candidate for cancer associated with abnormal expression of RNF20, if it has decreased in comparison with.
  • the third step instead of measuring expression in the SREBP1c, or in addition to measuring expression in SREBP1c, measuring the expression of a protein or a gene involved in lipid biosynthesis in which expression is promoted by the SREBP1c is measured. It may include a step. In this case, if the expression of the gene or protein involved in the lipid biosynthesis in the cell contacted with the test substance in the fourth step is reduced compared to the control cell not contacted with the test substance, the candidate substance is selected.
  • a first step of providing a cell with reduced or lacking expression of RNF20 A second step of treating said cells with a test substance that is expected to increase the expression of RNF20 or inhibit SREBP1c expression; A third step of measuring expression of PTTG1 in cells treated with the test substance; And comparing the PTTG1 expression in the cells contacted with the test substance and the control cells not in contact with the test substance, and when the expression of the PTTG1 in the cell contacted with the test substance decreased compared with the PTTG1 expression of the control. And a fourth step of selecting a candidate for treating a kidney cancer.
  • the third step measuring the expression of a protein or gene thereof involved in lipid biosynthesis that is promoted by SREBP1c
  • the fourth step Comparing the expression of the gene or protein involved in the lipid biosynthesis in the contacted cells and control cells not in contact with the test substance, the expression of the gene or protein involved in the lipid biosynthesis in the cell contacted with the test substance is If it is reduced compared to the expression of a gene or protein that promotes lipid biosynthesis, the step of selecting it as a candidate for the treatment of kidney cancer.
  • the present invention provides a method for detecting a prognosis of a RNF20 biomarker from a biological sample from a test subject to provide information necessary for the diagnosis or prognosis of renal or liver cancer; Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And associating with a diagnosis or survival prognosis of kidney or liver cancer of the subject when there is a change in the nucleic acid or protein levels of the subject-derived sample compared to the control sample. Or a diagnosis or prognosis for renal or liver cancer.
  • the present disclosure provides RNF20-SREBP1c-PTTG1 signaling in kidney cancer cells in vitro, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene.
  • RNF20 ring finger protein 20
  • the present application provides a RNF20-SREBP1c-lipid biosynthesis pathway of renal cancer cells in vitro, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene.
  • RNF20 ring finger protein 20
  • Kits according to the invention can be used to modulate RNF20-SREBP1c-PTTG1, or RNF20-SREBP1c-lipid biosynthesis signaling in vitro for various purposes.
  • RNF20 according to the present application can be usefully used as a therapeutic agent and diagnostic marker for kidney cancer, and the new molecular mechanisms RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipid biosynthesis pathways identified herein can be usefully used to find a renal cancer therapeutic agent. have.
  • RNA-Seq database was obtained from TCGA. ## P ⁇ 0.01 compared to normal kidney tissue; ### P ⁇ 0.001 Compared with normal kidney tissue.
  • E Immunohistochemical staining pictures of tumors and normal kidney tissue near the tumor of the same kidney cancer patient are shown. RNF20 is a representative tissue photograph detected. Criteria represent 100 ⁇ m.
  • F The change in RNF20 mRNA expression according to the presence or absence of RG108 treatment in the ACHN kidney cancer cell line (+; 250 ⁇ M or ++; 1 mM) was measured by qRT-PCR. RNF20 mRNA expression level was corrected by Cyclophilin mRNA expression level. RNF20 mRNA expression was expressed relative to the control. The data represent the mean ⁇ standard deviation of three independent samples.
  • H and I Survival curve analysis results according to the VHL mutation status.
  • J After the infection of ACHN and A498 kidney cancer cell lines with GFP (Mock) or RNF20 expressing adenovirus, the cell proliferation rate was measured using CCK-8 technique. It was observed that RNF20 inhibited the proliferation of renal cancer cells by overexpression. The results represent the mean ⁇ standard deviation of five independent samples. CCK-8, Cell Counting Kit-8.
  • K Cell growth curves were measured by CCK-8 technique after treatment of ACHN and A498 kidney cancer cell lines with nonspecific control siRNA (siControl) or RNF20 specific siRNA (siRNF20). Inhibition of RNF20 increased the proliferation of renal cancer cells. * P ⁇ 0.05, ** P ⁇ 0.01.
  • Figure 2 shows that SREBP1 and liposynthetic genes are increased in the tumor tissue of kidney cancer patients, which is inversely correlated with the expression level of RNF20.
  • A As a result of qRT-PCR analysis of SREBP1c in tumors and normal kidney tissue of the same kidney cancer patient, it was observed that the amount of SREBP1c mRNA expression was increased in tumor tissues compared to normal kidney tissue of the same patient.
  • B Analysis of TCGA RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed increased expression levels of SREBP1 in tumor tissues of kidney cancer patients.
  • C Analysis of SREBP1 expression according to the T stage of kidney cancer patients. RNA-Seq database was obtained from TCGA.
  • FIG. 3 shows that RNF20 inhibits SREBP1 inhibits the metabolic biosynthesis and renal cancer cell proliferation.
  • A ACHN kidney cancer cell lines were infected with adenoviruses expressing Myc-RNF20 or Flag-SREBP1c. Cell lysates were separated via SDS-PAGE and Western blotting was performed using antibodies. Validation of overexpression of RNF20 and SREBP1c.
  • B RNF20 or SREBP1c were continuously overexpressed using lentiviral in ACHN kidney cancer cell lines. Relative mRNA expression was measured by qRT-PCR. MRNA amount of each gene was corrected by mRNA expression amount of GAPDH gene. Each mRNA expression amount was expressed relative to the mock control.
  • Figure 4 shows the identification of PTTG1 as a new target gene of SREBP1c.
  • A Scatter plots of transcriptomes analyzed by RNA-Seq in livers of normal mice and mice lacking SREBP1c were expressed.
  • B PTTG1 mRNA expression levels in ACHN kidney cancer cell lines overexpressing RNF20 or SREBP1c were analyzed. PTTG1 mRNA expression was corrected by GAPDH and expressed as relative to the control.
  • C After transducing siRNF20 or siSREBP1 to ACHN kidney cancer cell lines, the amount of PTTG1 mRNA was analyzed by qRT-PCR.
  • PTTG1 mRNA expression level was corrected by GAPDH, and all mRNA expression levels were expressed relative to the control group.
  • siControl and siRNF20 were transduced into ACHN kidney cancer cells, and Western blotting was performed using antibodies. Inhibition of RNF20 increased SREBP1 and PTTG1 at protein levels. nSREBP1, nuclear SREBP1.
  • E ACHN kidney cancer cell lines were infected with SREBP1c adenovirus and transduced PTTG1 siRNA. Western blotting was performed using the antibody. It was observed that the amount of PTTG1 protein was increased by SREBP1c overexpression.
  • the HEK293 cell line was transduced with a luciferase reporter plasmid cloned with a PTTG1 promoter and a beta-galactosidase, RNF20, or SREBP1c expression vector. Cell lysates were analyzed by luciferase and beta-galactidase techniques. The data represent the mean ⁇ standard deviation of three independent samples. RLU, relative fluorescence (G) The qRT-PCR analysis of PTTG1 in tumors and normal kidney tissues of the same kidney cancer patients showed that PTTG1 mRNA expression was increased in kidney cancer tumor tissues.
  • RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed increased expression of PTTG1 in tumor tissues of kidney cancer patients.
  • PTTG1 expression level according to T stage of kidney cancer patients was analyzed.
  • RNA-Seq database was obtained from TCGA. ### P ⁇ 0.001 compared with normal height; ### P ⁇ 0.001 Compared with normal height.
  • J The correlation between RNF20 and PTTG1 mRNA expression in the TCGA database was calculated using Pearson's correlation analysis. Kaplan-Meier survival curves were analyzed in the group divided by the number of patients (n), Pearson's correlation coefficient (r), and P value (P)
  • K PTTG1 expression. P values were calculated via log-rank test. The survival prognosis of renal cancer patients with high PTTG1 expression was observed poorly.
  • FIG. 5 shows that betulin, an SREBP inhibitor, inhibits kidney cancer cell proliferation.
  • A ACHN and A498 kidney cancer cell lines were treated with various concentrations of betulin for 12 hours, and then cell lysates were separated by SDS-PAGE to perform western blotting. It was observed that betulin reduced the amount of SREBP1, fat metabolism biosynthesis, and cell cycle regulatory proteins. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1.
  • B and C ACHN and A498 kidney cancer cell lines were treated with betulin and cell proliferation was measured via CCK-8 technique. Betulin treatment inhibited renal cancer cell proliferation. The data represent the mean ⁇ standard deviation of five independent samples.
  • FIG. 6 shows that SREBP1c affects cell growth in kidney cancer by regulating cell cycle and fat metabolism.
  • A An experimental design schematic for verifying the inhibitory effect of PTTG1 or FASN inhibition through siRNA and the fat biosynthesis gene (FASN, ACC) through the drug is shown.
  • B and C PTRE1 siRNA was transduced into SREBP1c overexpressing ACHN kidney cancer cell line via lentiviral. After 48 hours, mRNA expression of adipose biosynthesis and cell cycle regulators was measured by qRT-PCR and corrected by GAPDH mRNA expression. The data represent the mean ⁇ standard deviation of three independent samples.
  • D Relative proliferation of the cells described in (B and C) was measured by CCK-8 technique.
  • ACHN kidney cancer cell lines were treated with ACC inhibitor Topa (TOFA, 10 ⁇ g / ml) or FASN inhibitor C75 (10 ⁇ g / ml) for 24 hours and then the amount of triglycerides in the cells was measured. The effect of fat metabolism reduction by inhibiting fat biosynthesis was verified.
  • (F) ACHN kidney cancer cell lines were treated with topa (10 ⁇ g / ml) or C75 (10 ⁇ g / ml) for 24 hours and then PTTG1 mRNA expression was measured by qRT-PCR. mRNA amount was expressed relative to the control. The amount of PTTG1 mRNA expression did not change by the control of fat biosynthesis.
  • FIG. 7 shows that RNF20 overexpression inhibits tumor proliferation in xenograft mice.
  • A BALC / c nude mice were injected subcutaneously with Mock or RNF20 overexpressing ACHN kidney cancer cells to generate tumors. Each group represents 10 tumors from five mice. Representative photographs showing tumor size at the end of the experiment show the effect of RNF20 overexpression on xenograft tumor growth in an individual. Validation of anticancer effect by RNF20 overexpression in vivo. Criteria represent 10 mm.
  • B Xenograft tumor volume of ACHN kidney cancer cells following RNF20 overexpression was measured over 35 days. The plot shows the mean volume plus standard error.
  • C Tumor weights were measured and expressed at the end of the experiment.
  • ACHN xenograft tumor sections overexpressing Mock or RNF20 were stained with hematoxylin eosin (H / E) or Oil red O and representative pictures were taken. Xenograft tumor sections were stained with Ki67 and TUNEL. TUNEL, terminal deoxynucleotide transferase dUTP niche terminal label. RNF20 overexpression demonstrated the effects of tumor growth inhibition and fat metabolism accumulation. Standard means 100 ⁇ m.
  • FIG. 8 shows a model (summary model) suggesting SREBP1-dependent adipose biosynthesis of RNF20 and renal tumor suppression function through cell cycle control in kidney cancer.
  • Decreased expression of RNF20 and increased expression of SREBP1 in kidney cancer patients are associated with poor survival prognosis.
  • RNF20 inhibition leads to the activation of SREBP1 leading to renal cancer oncogenesis.
  • SREBP1 regulates the cell cycle through a new target gene, PTTG1.
  • Betulin, a SREBP1 inhibitor also inhibits renal cancer cell proliferation through adipose biosynthesis control and G1 cell cycle arrest. Taken together, reduced RNF20 expression promotes tumorigenicity of renal cancer through SREBP1-dependent adipose biosynthesis and cell proliferation activity.
  • RNF20 shows tumor suppression function in kidney cancer cells, but does not affect cell proliferation of normal kidney cells.
  • A Western blotting technique for the expression of RNF20 protein in human primary renal cortical epithelial (HRCE) cell lines, normal kidney cell lines such as HEK293, and kidney cancer cell lines such as ACHN, A498, and Caki-2. Analyzed.
  • B RG108 (+; 250 ⁇ M or ++; 1 mM) was treated in HEK293 cell line for 48 hours, and RNF20 mRNA expression was measured by qRT-PCR. The expression level of RNF20 mRNA was corrected by the expression level of Cyclophilin mRNA and expressed as a relative value to the control group.
  • the data represent the mean ⁇ standard deviation of three independent samples. n.s., not significant.
  • C ACHN and A498 kidney cancer cell lines were infected with GFP (Mock) or Myc-RNF20 expressing adenovirus. After 24 hours, the cell lysate was separated by SDS-PAGE, followed by Western blotting using an antibody.
  • D After transducing siControl and siRNF20 into ACHN kidney cancer cell lines, RNF20 expression was measured by Western blotting.
  • E HRCE and HEK293 cell lines were infected with Mock or Myc-RNF20 expressing adenovirus and cell lysates were isolated via SDS-PAGE, followed by Western blotting using antibodies.
  • Figure 10 shows that the expression of liposynthetic enzymes in renal cancer tumor tissue is increased and inversely correlated with RNF20 expression.
  • A qRT-PCR analysis of FASN in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient.
  • B shows the corrected FASN RNA-Seq expression in tumors and normal kidney tissue of kidney cancer patients.
  • C Analysis of FASN expression according to renal cancer T stage.
  • D qRT-PCR analysis of SCD1 in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient.
  • E Corrected SCD1 RNA-seq expression levels are shown in renal cancer tumors and normal kidney tissues.
  • FIG. 11 shows that RNF20 inhibition in kidney cancer cells promotes lipobiosynthesis and cell proliferation.
  • siRNF20 or siSREBP1 were transduced into ACHN kidney cancer cell lines. After 48 hours, cell lysates were separated on SDS-PAGE and Western blotting was performed using antibodies. nSREBp1, nuclear SREBP1.
  • B and C After knocking down RNF20 or SREBP1 by siRNA in ACHN kidney cancer cell lines, mRNA expression levels of adipose biosynthesis-related genes were analyzed using qRT-PCR. mRNA expression level was corrected by GAPDH mRNA expression level and expressed as relative value to Mock control group.
  • Figure 12 shows that PTTG1 is induced by SREBP1c, the expression level is increased in the tumor tissue of kidney cancer patients.
  • Kidney, liver and adipose tissues of normal mice and mice lacking SREBP1c were isolated. After extracting RNA from various tissues, mRNA expression levels of SREBP1c and PTTG1 were analyzed by qRT-PCR. mRNA expression level was corrected by TATA-binding protein (TBP) expression level.
  • TBP TATA-binding protein
  • EAT visceral adipose tissue, IAT; Subcutaneous adipose tissue; BAT; Brown adipose tissue ** P ⁇ 0.01, *** P ⁇ 0.001.
  • B SRE motif and E-BOX sequence present in PTTG promoter in various species.
  • FIG. 13 shows that betulin effectively lowers kidney cancer cell proliferation.
  • siControl and siRNF20 were transduced into ACHN kidney cancer cell lines. After 24 hours of betulin treatment, qRT-PCR was used to measure mRNA expression of adipose biosynthesis and cell cycle regulators. mRNA expression level was corrected by GAPDH expression level. The data represent the mean ⁇ standard deviation of three independent samples. # P ⁇ 0.05 compared with control, ## P ⁇ 0.01 compared with control, ### P ⁇ 0.001 compared with control, ** P ⁇ 0.01, *** P ⁇ 0.001. Growth curves of kidney cancer cell lines described in (C and D) (A and B) were measured using the CCK-8 technique. P ⁇ 0.05, *** P ⁇ 0.001, ns, not significant; CCK-8, Cell Counting Kit-8.
  • Figure 14 shows that SREBP1c regulates renal cancer cell proliferation through two pathways (lipid biosynthesis and cell cycle regulation).
  • a and B ACHN kidney cancer cell lines were treated with Topa (10 ⁇ g / ml) or C75 (10 ⁇ g / ml) for 24 hours. MRNA expression of adipose biosynthesis and cell cycle regulators was corrected by GAPDH mRNA expression and expressed as relative to control.
  • C The growth rate of the cells described in (A and B) was measured by CCK-8 technique.
  • RNF20 controls apoptosis and apoptosis genes in kidney cancer xenograft tumors.
  • the effect of RNF20 overexpression on apoptotic gene expression control in ACHN kidney cancer xenograft tumors was analyzed using qRT-PCR. Relative mRNA expression was corrected by GAPDH mRNA expression and expressed as relative to the mock control. ** P ⁇ 0.01, *** P ⁇ 0.001.
  • FIG. 16 shows the relative expression levels of RNF20 and SREBP in HCC, Huh-7 and HepG2 cell lines, which are liver cancer cell lines, as measured by gene (left) and protein (level), and compared with the control group (HEK293T) in liver cancer cell line. The amount of expression is decreased, and the SREBP is increased.
  • HEK293T human embroynic kidney cell; non-cancer cell
  • Huh-7 human hepatocellular carcinoma cell; mutant p53-Y220C
  • HepG2 human hepatoblastoma cell; wild-type p53. * P ⁇ 0.05 vs. HEK293T, ** P ⁇ 0.01 vs. HEK293T.
  • FIG. 17 shows that cell proliferation was reduced in hepatocellular carcinoma cell lines by RNF20 expression.
  • the cell lines were measured using CCK-8 (cell counting kit-8) 48 hours after transfer to plasmid expressing RNF20.
  • FIG. 18 is a Western blot showing the decrease of the concentration of SREBP1c protein in liver cancer cell line by RNF20 expression. Analysis was performed 48 hours after delivery of the cell line to the RNF20 expressing plasmid.
  • Figure 19 is a quantitative RT-PCR results showing that the expression of adipogenetic genes in liver cancer cell lines by RNF20 expression, the analysis was performed 48 hours after delivery of the cell line to the RNF20 expressing plasmid. * P ⁇ 0.05 vs. Mock, # P ⁇ 0.05.
  • FIG. 21 shows that SREBP1 protein concentration was increased in liver cancer cell lines which inhibited the expression of RNF20 using siRNF20. Analysis was performed 72 hours after treatment with siRNF20.
  • Figure 22 is a quantitative RT-PCR results showing the increase in the expression of adipogenic genes in hepatocarcinoma cell lines that inhibited the expression of RNF20 using siRNF20, the analysis was performed 72 hours after treatment with the cell line siRNF20. * P ⁇ 0.05, ** P ⁇ 0.01.
  • the present application shows that the expression level of ring finger protein 20 (RNF20) is decreased in tumor tissues of renal and liver cancer patients, and the overexpression of RNF20 is associated with the proliferation and fat metabolism activity of liver and kidney cancer cells in liver or kidney cancer cell lines and xenograft models.
  • RNF20 as a key regulator, it was found to regulate the RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipid biosynthesis pathway.
  • the present application for the prevention or treatment of kidney cancer or liver cancer including a ring finger protein 20 (RNF20) gene or a protein or a functional equivalent thereof, or a substance that increases the expression through methylation inhibition of the RNF20 gene To a composition.
  • RNF20 ring finger protein 20
  • Ring finger protein 20 included in the composition according to the present invention is a type of E3 ubiquitin ligase that plays a variety of roles in transcriptional regulation, DNA damage response, stem cell differentiation and lipogenesis, and contributes to histone remodeling. Promote monoubiquitination of H2B (Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10 , 483-488; Shema et al., 2011, supra).
  • RNF20 included in the composition according to the present invention can be used in various forms and derived so long as the effect according to the present application is achieved.
  • RNF20 or a functionally equivalent variant from mammals, in particular humans is used, the genes and protein sequences of which are known as NCBI Entrez Gene ID: 56254 and 34878777, respectively.
  • SEQ ID NO: 1 and 2 respectively, the RNF gene and protein sequence is used.
  • Genomic DNA and cDNA can be prepared according to methods known in the art.
  • Genomic DNA for example, extracts genomic DNA from cells having the gene of interest and constructs a genomic library (vectors may be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and viewed the library.
  • Colony or plaque hybridization is performed using a probe made on the basis of DNA encoding the protein of the invention (eg, SEQ ID NO: 2), or DNA encoding the protein of the present invention (eg, SEQ ID NO: 2).
  • cDNA synthesizes a cDNA based on mRNA extracted from a cell having an RNF20 gene, inserts the synthesized cDNA into a vector such as ⁇ ZAP, prepares a cDNA library, and expands the cDNA library.
  • a vector such as ⁇ ZAP
  • cDNA library can be prepared by colony or plaque hybridization or by PCR.
  • the term "functionally equivalent variant” refers to a variant compared to a wild type protein or gene sequence thereof derived from a particular species, but having the efficacy identified herein.
  • the RNF20 protein of the present invention or a gene encoding the same may be a mutants encoding a protein derived from a human sequence consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, Derivatives, alleles, variants and homologues.
  • variations in nucleotide sequences may not be accompanied by variations in amino acids in proteins (degeneracy variants), and such degeneracy mutants are also included in the gene of the present invention.
  • the RNF20 protein and the gene encoding the functionally equivalent protein are known in the art, for example PCR methods (Saiki et al., Science, 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988).
  • those skilled in the art will be able to isolate primers from a variety of mammals having high homology with the RNF20 gene by designing primers capable of specific hybridization thereof from known RNF20 gene sequences.
  • the gene isolated as described above has high homology with the amino acid sequence of the human-derived RNF20 protein at the amino acid level.
  • High homology refers to the identity of at least 50%, more preferably at least 70%, more preferably at least 90% (eg, at least 95%) sequences throughout an amino acid sequence.
  • the homology of amino acid sequences or nucleotide sequences is based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993), a program called BLASTN or BLASTX (Altschul et al, J. Mol. Biol. , 215, 403-410, 1990) and specific methods are known on the following website (http: //www,ncbi.nlm.nih.gov.).
  • the RNF20 protein or gene included in the composition according to the present invention includes all of full length or truncated forms.
  • sequence variations according to a specific individual, region, environment, etc. even if they are derived from the same host, for example, humans, and of course, some sequences have been modified (deleted, substituted, added), but all functionally equivalent variants are present. It can be used in the present invention. In one embodiment it is of human origin and is as previously mentioned.
  • the gene encoding the protein is RNF20 in a target cell, for example, a tumor cell of mammalian origin, in particular a kidney cancer cell, according to known methods such as recombinant DNA technology.
  • a target cell for example, a tumor cell of mammalian origin, in particular a kidney cancer cell
  • the gene may be introduced and used as a linear DNA, a plasmid vector or other DNA delivery vector.
  • Such vectors are preferably non-viral and are not particularly limited as long as they can express the RNF20 gene in eukaryotic cells, and reference may be made to those described in the Examples herein.
  • the RNF20 gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, for example, a viral vector, and then included in a viral particle as a carrier according to a known method.
  • the viral vector is not limited as long as the protein of the present invention can be introduced into a desired cell or tissue, and is preferably an adenovirus vector, and the viral vector is capable of expressing a gene contained therein in a eukaryotic cell. It is not particularly limited.
  • the vector may be purified and used after transfection into an appropriate cell in a cell culture system according to a known method.
  • proteins of the invention include, for example, purified proteins, water soluble proteins, or those in fusion with protein or amino acid residues in a form bound to a carrier for delivery or administration to a target cell.
  • Substances that increase the expression of RNF20 included in the composition according to the present application is a substance that inhibits methylation of the promoter region of the gene, particularly an endogenous gene, that is, the cell itself, in particular, a substance that inhibits methylation in CpG islands. Inhibition of methylation promotes the transcription of RNF20, resulting in increased expression of RNF20 in cells. Therefore, various materials which exhibit this effect can be included in the compositions according to the present application. In one embodiment a DNA methyltransferase (DNMT) inhibitor is used.
  • DNMT DNA methyltransferase
  • DNA methyltransferase (DNMT) inhibitors include, but are not limited to, nucleoside analogs as epigenetic gene-modulating drugs such as, for example, azacytidine, decitabine, and zebularine, and may include various substances including the effects described above.
  • Non-nucleoside analogs may also be used, including hydralazine, RG109, procainnamide, procaine, SCI-1027, and the like.
  • Non-nucleoside analogs interfere with the binding of DNMT to the CpG island, which in turn inhibits methylation, and in the process specifically interacts with DNMT.
  • RG108 is used.
  • Kidney cancer in which the composition according to the present invention is used includes renal cell carcinoma that occurs in the parenchyma of the kidney (consisting of the medulla and the cortex as a collection of urine-forming cells in the kidney), and clear cell type renal cell carcinoma (clear cell cancer) RCC, ccRCC), Papillary RCC, Chromophobe RCC, Medullary RCC, Unclassified RCC, Kidney transitional cancer cell carcinoma (TCC), renal eosinophilic granuloma (ranal oncocytoma). In one embodiment, it is ccRCC.
  • HCC hepatocellular carcinoma
  • composition according to the present invention exhibits an effect irrespective of von Hippel-Lindau (VHL) variation or condition of kidney cancer, and thus can be used for the treatment and diagnosis of kidney cancer of more various causes.
  • VHL von Hippel-Lindau
  • compositions and methods herein can be used for the treatment and diagnosis of kidney cancer independently of VHL mutations.
  • the kidney cancer in which the compositions and methods herein are used may or may not include VHL variations.
  • the compositions and methods herein can be used in kidney cancer that is not caused by VHL mutations.
  • treatment is a concept that includes inhibiting, eliminating, alleviating, alleviating, ameliorating, and / or preventing a disease or symptom or condition resulting from the disease.
  • composition of the present application may further contain a compound which maintains / increases the solubility and / or the absorbency of at least one active ingredient or an active ingredient exhibiting the same or similar function in addition to RNF20.
  • pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of kidney cancer.
  • composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, seeding, and one or more of these components, as necessary.
  • Other conventional additives such as buffers and bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs.
  • Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used.
  • it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.
  • the method of administration of the composition of the present application is not particularly limited thereto, and known administration methods may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) or oral, depending on the desired method.
  • parenteral administration for example, intravenous, subcutaneous, intraperitoneal, or topical
  • it can be administered through a patch-type, nasal / respirator attached to the skin, and administration by intravenous injection is preferable to obtain a rapid therapeutic effect.
  • the dosage may vary widely depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient.
  • Parenteral administration may be preferred for protein preparations comprising the RNF20 gene or polypeptide, but does not exclude other routes and means.
  • dosage units include, for example, about 0.01 mg to 100 mg but do not exclude the below and above ranges.
  • the daily dose may be about 1 ⁇ g to 10 g, and may be administered once to several times
  • the present application relates to the use of RNF20 as a biomarker, and more particularly to a composition or kit for diagnosing or prognosticting liver or kidney cancer comprising a substance for detection thereof.
  • diagnosis refers to determining the susceptibility of a subject's disease to a particular disease or condition, determining whether he or she currently has a particular disease or condition, prognosis of the subject having a particular disease or condition ( prognosis (eg, identifying a transitional cancer state, determining stage or progression of a cancer, or determining the responsiveness of a cancer to treatment) or to determine therametrics (eg, to provide information about treatment efficacy). Monitoring the state of an object).
  • prognosis eg, identifying a transitional cancer state, determining stage or progression of a cancer, or determining the responsiveness of a cancer to treatment
  • therametrics eg, to provide information about treatment efficacy
  • diagnostic biomarker or diagnostic marker is a substance that can diagnose cancer-causing tissues or cells from normal cells or appropriately treated tissues or cells and diagnose the diseased tissues or regions as compared to normal samples.
  • Protein or nucleic acid showing an increase or decrease in Renal cancer diagnostic marker herein is an RNF20 protein or a nucleic acid or gene encoding the same that reduces expression in kidney cancer tissue.
  • Biological sample as used herein refers to a substance or mixture of substances that includes one or more components capable of detecting a biomarker and includes, but is not limited to, cells, tissues or body fluids, such as whole blood, plasma, and serum from an organism, in particular humans. It is not. It also includes cells or tissues derived directly from an organism as well as cultured in vitro. Various samples may be used for the detection of liver or kidney cancer markers according to the present disclosure, but are not limited thereto. In one embodiment, whole blood, serum and / or plasma may be used. In other embodiments, renal tissue / cells or in vitro cell cultures obtained from, or suspected of developing or likely to develop, liver cancer or kidney cancer may be used. It also includes fractions or derivatives of the blood, cells or tissues. When using a cell or tissue, the cell itself or a fusion of the cell or tissue may be used.
  • RNF20 protein and nucleic acid sequences thereof are known herein and are also as previously mentioned, but not limited to, including functional equivalents thereof.
  • Detection herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level detection. Such methods are well known in the art and those skilled in the art will select appropriate methods for carrying out the present application. Can be.
  • the markers according to the invention can be detected at the level of the detection of the presence of nucleic acids, in particular of mRNA and / or protein, and / or their expression levels themselves, changes in expression levels, differences in expression amounts, through quantitative or qualitative analysis.
  • Such markers for diagnosing liver or kidney cancer according to the present application are based on their functional and / or antigenic characteristics.
  • the activity, function or activity of a protein can be detected using agents that specifically interact at the nucleic acid encoding the protein, particularly at the mRNA and / or protein level.
  • the present application also includes an antibody or aptamer specifically recognizing a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence, or a protein encoded by the nucleic acid sequence. It relates to a marker for diagnosing kidney cancer.
  • detecting substance is a substance capable of detecting the marker of the present invention quantitatively or for the presence of a protein or nucleic acid such as mRNA level.
  • Methods for the analysis of the amount and presence of the protein expression patterns are known, for example Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, oukterroni Ouchterlony) immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like.
  • Reagents for detecting protein or nucleic acid levels are known, for example, the former may be an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor that specifically interacts with the marker or It can be detected through reaction with a ligand or cofactor, or a mass spectrometer can be used.
  • Reagents or materials that specifically interact with or bind to the markers of the present disclosure may be used with chip or nanoparticles.
  • the diagnostic composition according to the present disclosure may comprise reagents necessary for the detection of an RNF20 marker at a protein or nucleic acid such as at the mRNA level.
  • reagents detectable at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands, cofactors, and the like. Such reagents can be incorporated into nanoparticles or chips as needed. Proteins can also be detected using mass spectrometry.
  • RNA levels, expression levels or patterns using transcription polymerase chain reaction (RT-PCR) / polymerase chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assays, chips or Northern blots Methods can be used, and such assays are known and can also be performed using commercial kits, and one skilled in the art will be able to select the appropriate one for the practice herein.
  • a detection reagent in a method for measuring the presence and amount or pattern of the mRNA by RT-PCR for example, a primer specific for mRNA of the marker of the present application is included.
  • primer is meant a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template.
  • the detection reagent used herein may be conjugated with a coloring material such as a fluorescent material for signal detection.
  • Detection herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level detection. Such methods are well known in the art, and those skilled in the art will recognize methods suitable for the practice of the present application. You will be able to choose.
  • the original detection reagent comprises an antibody
  • the RNF20 protein detection of the present application is carried out using a monoclonal antibody that specifically binds thereto.
  • Antibodies that can be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies.
  • Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)).
  • fusion methods Kellow and Milstein, European Journal of Immunology, 6: 511-519 (1976)
  • recombinant DNA methods US Pat. No. 4,816,56
  • the immunoassay format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunity. Including but not limited to fluorescent staining and immunoaffinity purification.
  • the immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T.
  • the detection reagent is a reagent for RNA analysis
  • the detection according to the present application is carried out at the mRNA level.
  • mRNA detection is usually carried out by Northern blot or reverse transcriptase PCR (polymerase chain reaction).
  • the RNA of a sample is specifically isolated from mRNA, and then cDNA is synthesized therefrom, and then a specific gene or a combination of primers and probes is used to detect a specific gene in the sample. Or it is a method which can determine the expression amount.
  • the present application provides a therapeutic agent for cancer associated with abnormal expression of RNF20 targeting RNF20-SREBP1c (Sterol Regulatory Element Binding Transcription Factor 1c) -PTTG1 (Pituitary Tumor Transforming 1) or RNF20-SREBP1c-lipidogenesis promoting protein signaling pathway. It relates to a screening method.
  • the low expression or suppression or lack of expression of RNF20 is associated with the development of renal cancer or liver cancer, and elucidated the mechanism of the various cancers, if abnormal expression of RNF20, in particular, low expression or lack of, led to the development of cancer.
  • Therapeutic agents for can be discovered by the screening methods herein. Examples of such cancers are not particularly limited to those including kidney cancer or liver cancer.
  • kidney cancer, particularly kidney cancer that is not caused by von Hippel-Lindau (VHL) gene mutations, as described above, may or may not include VHL mutations and includes VHL mutations. Even if these mutations are not the cause of kidney cancer.
  • VHL von Hippel-Lindau
  • the method according to the present invention comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase expression of RNF20; A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And comparing the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance and the control cells not in contact with the test substance, so that the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance is less than that of the control. And a fourth step of selecting it as a therapeutic agent candidate for cancer associated with abnormal expression of RNF20.
  • the reduction or lack of expression of RNF20 is reduced or inhibited by the mutation or epigenetic regulation of the RNF20 gene itself, or the expressed protein is epigenetic even if it does not involve abnormality of the gene.
  • the desired function is not performed by regulation or the like, those skilled in the art will be able to determine the extent to which expression is reduced or inhibited with reference to normal cells and those described in the Examples herein. For example, if gene expression (or transcription) of RNF20 is inhibited, leading to a decrease in protein expression (or translation), or if it is expressed as a protein but the protein does not function as desired due to gene mutation, the gene is methylated. And when the expression of the gene is suppressed. Examples of cases lacking include the case where the gene of RNF20 is present but not expressed by epigenetic regulation such as methylation.
  • cells or cell lines derived from renal or hepatic cancer tissue are used as the cells with reduced or lacking the expression of RNF20.
  • Expression of RNF20 refers to a decrease or lack in gene and / or protein levels, wherein a decrease or lack of expression is reduced in comparison to using a normal kidney cell line as a control. Based on the level, the reduced level can be judged.
  • the cells with reduced expression of RNF20 used in the methods according to the present disclosure include, but are not limited to, ACHN, A498, or Caki-2 as a kidney cell line, Huh-7 or HepG2 as a liver cancer cell line.
  • the method comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating with a test substance expected to increase the expression of RNF20 or inhibit SREBP1c expression of said cells; A third step of measuring expression of PTTG1 in cells treated with the test substance; And comparing the PTTG1 expression in the cells contacted with the test substance and the control cells not in contact with the test substance, and when the expression of the PTTG1 in the cell contacted with the test substance decreased compared with the PTTG1 expression of the control. And a fourth step of selecting a candidate for treating a kidney cancer.
  • the candidate material in the third step, instead of or in addition to measuring the expression of SREBP1c, measuring the expression of a gene or a protein thereof involved in lipid biosynthesis in which expression is promoted by the SREBP1c.
  • the candidate material in the fourth step, has a decreased expression of a gene or protein involved in the lipid biosynthesis in comparison with control cells not in contact with the test material in cells in contact with the test material.
  • the third step measuring the expression of a protein and / or a gene involved in lipid biosynthesis in which expression is increased, enhanced, or promoted by the SREBP1c
  • the fourth step instead, the expression of the proteins and / or genes involved in the lipid biosynthesis in the cells in contact with the test substance and in control cells not in contact with the test substance was compared to determine the lipid biosynthesis in the cells in contact with the test substance. If the expression of the involved protein is reduced compared to the expression of the protein involved in the lipid biosynthesis of the control group comprising the step of selecting it as a candidate for the treatment of kidney cancer.
  • RNF20-SREBP1c-lipid biosynthesis promoting pathway was found, wherein the gene or the protein involved in lipid biosynthesis is that its transcription is increased by SREBP1c and its expression to the protein is increased.
  • the increase increases or promotes the synthesis of lipids in the cell. Therefore, as long as these characteristics are not particularly limited, substances that are involved in lipid metabolism in kidney cancer are FAS (Fatty acid synthase, or gene is FASN), SCD1 (Stearoyl-CoA desaturase-1), or ELOVL6 (Elongation of very Long chain fatty acids protein 6) can be selected by measuring the expression of the gene or protein.
  • each protein and its gene sequence are for example FASN NCBI gene ID (2194), protein DB (41872631); ACC1 Gene ID (31), Protein DB (38679960); SCD1 gene ID (6319), protein DB (53759151); And ELOVL6 Gene ID (79071), Protein DB (195539343), will be readily apparent to those skilled in the art, based on the sequences and disclosed herein, the expression of the protein or gene. It is also not intended to exclude the measurement of other genes known as target genes of SREBP1c that are involved in lipid biosynthesis in addition to these genes.
  • the expression of the protein involved in the lipid metabolism in the cell contacted with the test substance decreases compared with the expression of the protein involved in the lipid metabolism of the control group, it may be selected as a candidate for treating cancer of the kidney.
  • the molecular targets are RNF20 and / or SREBP1c, and the substance which increases the expression of the former or the substance which decreases the expression of the latter can be used as a test substance for screening a therapeutic agent for kidney cancer.
  • the test substance used in the method of the present invention is a substance which is expected to modulate the expression and / or activity of the target gene or protein described above.
  • the compound may have a low molecular weight therapeutic effect.
  • compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used.
  • such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions.
  • Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely illustrative. It is not limited to this.
  • PTTG1 has disclosed its sequence, and may refer to GenBank ID: NM_004219 for human cDNA sequence and NP_001269311 for protein sequence. Expression of PTTG1 can be detected at the protein or gene level, for which reference can be made above.
  • Candidates compared the PTTG1 expression in cells in contact with the test substance and control cells not in contact with the test substance, whereby the expression of the PTTG1 in the cells in contact with the test substance decreased compared to the PTTG1 expression of the control. If this can be selected as a candidate candidate for the treatment of kidney cancer.
  • increasing the expression of RNF20 or suppressing SREBP1c expression by the treatment of the test substance can be determined by measuring the expression of proteins involved in lipid metabolism in the cells.
  • the type of cell used in the present method and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount.
  • a substance which results in a decrease in the expression or activity of the protein in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance.
  • the present disclosure provides a method for detecting a cancer or kidney cancer, comprising: detecting a level of nucleic acid and / or protein of a RNF20 biomarker from a biological sample from a test subject to provide information necessary for the diagnosis or prognosis of liver or kidney cancer; Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And when there is a change in the nucleic acid or protein level of the subject-derived sample compared to the control sample, correlating with a renal cancer diagnosis or survival prognosis of the subject. .
  • Detection of the amount of expression of a marker used in the method according to the invention can be determined at the protein and / or mRNA expression level, as mentioned above.
  • Subject biological samples may also refer to the foregoing.
  • the control group is a normal control group, and when the level of the marker decreases in the subject compared to the level of the marker determined in the normal control group, the subject agent has a poor renal cancer or a poor survival prognosis.
  • Survival prognosis refers to 5-year survival after diagnosis of kidney cancer, which can be determined by combining pathological, clinical and molecular biological findings. Survival rates are especially associated with cancer stages, which are generally 88-100% in stage 1, 63-88% in stage 2, 34-59% in stage 3 and 0-20 in stage 4 %, Which means lower than that.
  • the step of associating a sample of the subject with a normal control group sets a threshold value for diagnosing the onset of each marker, and then detects the detection result of the subject with the threshold value. Can be compared. Threshold settings may refer to, for example, the methods described in the Examples herein.
  • non-protein clinical information i.e., non-marker clinical information
  • non-marker clinical information includes, but is not limited to, for example, one or more of age, sex, weight, diet, body mass, underlying disease, ultrasound, computed tomography (CT) of the patient.
  • CT computed tomography
  • the present disclosure provides RNF20-SREBP1c-PTTG1 signaling in cells using a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that results in increased expression of the gene in vitro or in an animal.
  • RNF20 ring finger protein 20
  • the present disclosure also provides RNF20-SREBP1c-lipid biosynthesis of cells using a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that results in increased expression of the gene in vitro or in an animal.
  • RNF20 ring finger protein 20
  • kits and methods and the regulatory mechanism using the same may be usefully used as treatments for kidney or liver cancer, existing drug tests, drug development, research tools, but are not limited thereto.
  • ACHN, A498, HEK293, Caki-2 and human primary renal cortical epithelial (HRCE) cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to the supplier's manual .
  • ATCC American Type Culture Collection
  • ACHN and A498 cell lines were cultured in Eagle's minimum essential medium (MEM) medium containing 10% fetal bovine serum (FBS), penicillin (100 U / ml) and streptomycin (100 ⁇ g / ml).
  • HEK293 and Caki-2 cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) medium containing 10% FBS, penicillin and streptomycin.
  • DMEM Dulbecco's modified Eagle medium
  • HRCE cell line 0.5% FBS, 10 nM triiodothyronine, 10 ng / ml epidermal growth factor, 100 ng / ml hydrocortisone, 5 ⁇ g / ml insulin, 1 ⁇ M epinephrine, 5 ⁇ g / ml transferrin, 2.4 mM L-alanine-L-glutamine Cultured in renal epidermal cell culture medium containing penicillin and streptomycin. All cells were cultured in 5% carbon dioxide, 37 °C conditions. Betulin and BODIPY 493/503 were purchased via Sigma-Aldrich. C75 and TOFA were purchased from Abcam. Propidium iodine compounds were purchased from BD Biosciences.
  • Tumor tissue and normal kidney tissue specimens of liver and kidney cancer patients were provided by Seoul National University Hospital. The study was approved by a review institution at Seoul National University Hospital (approved number: H-1501-011-636). Due to the retrospective nature of this study, the patient's consent was not required.
  • RNA sequencing results for 533 kidney cancer tumor tissues and 72 normal kidney samples were analyzed using the 1-99th percentile (bar), 25-75th percentile (box), and median (line within the box). Indicated. Patients were ranked according to gene expression from TCGA RNA-Seq data for survival analysis. Patients with higher than average gene expression were defined as "high” and the rest as "low”. Overall survival curves were estimated by Kaplan-Meier survival analysis and survival differences between the two groups were compared by log-rank analysis.
  • tissue microarray included 50 kidney cancer tumor tissue sections and 9 normal kidney tissues.
  • streptavidin-biotin complex technique was used to detect RNF20 and SREBP1, and each antibody was purchased from Abcam and BD Biosciences.
  • Adenovirus plasmids were constructed as reported in previous papers (1). Briefly, cDNAs encoding rat SREBP1c amino acids 1-403 and the entire mouse RNF20 were cloned into an AdTrack-CMV shuttle vector and constructed via an Ad-Easy adenovirus vector system. In all experiments, adenovirus encoding GFP was used as a control. Adenovirus was propagated in HEK293A cell line and purified using the previously reported CsCl density gradient centrifugal method (2).
  • RNF20 and SREBP1c cDNA were cloned into the lentiviral vector pLVX-EF1 ⁇ -AcGFP1-N1.
  • the lentiviral vector and pAX2 and pMD2.G vectors were transduced into HEK293T cell line using lipofectamine 2000. 48 hours after transduction, the virus was collected and filtered through a 0.45- ⁇ m filter. Thereafter, the cells were incubated for 18 hours in a culture medium containing 8 ⁇ g / ml polybrene and virus in an ACHN cell line. Infected cells were recovered for 48 hours and only colonies resistant to puromycin were selected for use in the experiment.
  • Cells and tissues were radioimmunoprecipitation assay containing 150 mM sodium chloride, 50 mM tris-sulfate, pH 7.4, 1% NP-40, 0.25% sodium dioxycholate, 1 mM EDTA, 1 mM PMSF, and a protease inhibitor mixture. Dissolved in (RIPA) buffer. Equal amounts of protein in each sample were separated on an SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was then added to TBS solution containing 0.1% Tween-20 to suppress nonspecific reactions by adding 5% nonfat milk or 3% bovine serum albumin.
  • PVDF polyvinylidene difluoride
  • Antibodies that bind -tag, Flag-tag or ⁇ -actin were bound to the PVDF membrane. After binding the horseradish peroxidase-conjugated secondary antibody to the PVDF membrane, the protein was visualized by chemiluminescence detection using a LuminoImager (LAS-3000) machine.
  • RNA was extracted with TRIzol lysis reagent.
  • CDNA was synthesized from the same amount of RNA using RevertAid reverse transcriptase.
  • Relative mRNA expression was measured by quantitative real-time PCR analysis (qRT-PCR) and calculated after correction with GAPDH or Cyclophilin mRNA.
  • SiRNA double strands of RNA20, SREBP1, PTTG1 and FASN were synthesized in Bioneer (South Korea). SiRNA transduction was performed to ACHN kidney cancer cell lines using the lipofectamine RNAiMAX system according to the provider instructions.
  • Cell proliferation was measured using a previously reported Cell Counting Kit-8 (CCK-8) system (3).
  • cell coloration curves were obtained by performing colorimetric analysis to detect metabolites of living cells.
  • ACHN cell lines with lentivirus overexpressing RNF20 or SREBP1c were cultured in 6-well plates. Cells were incubated at 5% carbon dioxide at 37 ° C. for 7 days, then fixed with formaldehyde and stained with crystal violet.
  • Trypsin-treated cells were washed with phosphate buffered saline (PBS) and fixed at 70 ° C. for 30 minutes at 4 ° C.
  • the fixed cells were washed twice with phosphate buffered saline, and then rinsed in a solution containing 0.1% nonniche P-40, 100 ⁇ g / ml RNA degrading enzyme and 2.5 ⁇ g / ml propidium iodide (PI) for 30 minutes.
  • Stained. Stained cells were analyzed by flow cytometry using the FACS Canto II machine, and the number of cells in each cell cycle was calculated using the ModFit LTTM cell cycle analysis program.
  • Intracellular triglycerides were measured by colorimetric analysis as previously reported and expressed in mg of lipid per mg of protein in cells (1). Briefly, the cell lysate was extracted using 5% Triton X-100, and then immersed in an 80 ° C. water bath and immersed in ice twice. After centrifugation at 12,000 rpm for 5 minutes, the supernatant was collected and the amount of intracellular triglyceride was measured using Infinity triglyceride assay. The measured value was corrected by the total protein amount analyzed by the BCA protein quantitative kit.
  • the ACHN kidney cancer cell line was incubated for 24 hours with or without 10 ⁇ M betulin, washed twice with phosphate buffered saline, and then fixed for 4 minutes with 4% paraformaldehyde.
  • the immobilized cells were washed twice with phosphate buffered saline containing Tween-20 and stained with fluorescein isothiocyanate (FITC) bound to bodiphy 493/503 for 1 hour in the dark. .
  • Samples were stained with a betashield solution containing 4 ′, 6-diamidino-2-phenylindole (DAPI) and observed using a Zeiss LSM 700 confocal microscope. All pictures were observed and analyzed under the same conditions.
  • DAPI 6-diamidino-2-phenylindole
  • PTTG1 promoter region (transcription start point -908 to +25 nucleotides) was cloned into pGL3-basic vector.
  • DNA plasmids were transduced into HEK293 cell line using the previously reported calcium-phosphate method (4). After 36 hours of incubation, the transduced cells were harvested and lysed using a buffer containing 25 mM tris-phosphate (pH 7.8), 10% glycerol, 2 mM EDTA, 2 mM DTT, and 1% Triton X-100. Was extracted. Luciferase and beta-galactidase activity were measured according to the instructions of the provider. Luciferase activity was corrected to beta-galactosidase activity.
  • mice Five weeks after transplantation, the mice were euthanized by inhalation of carbon dioxide and xenograft tumors were taken out and analyzed.
  • Xenograft tissue samples were fixed in 4% paraformaldehyde and placed in 30% sucrose and then in OCT solution.
  • Xenograft tumor tissue sections were subjected to hematoxylin / eosin (H / E) and oil red o staining and immunohistochemistry (5). Briefly, Ki67 protein and TUNEL techniques were performed using a streptavidin-biotin complex system. Photographs were observed using an EVOS ORIGINAL microscope and NIKON TMS inverted microscope.
  • Results were expressed as mean ⁇ standard deviation or mean ⁇ standard error ( Figures 7B and 7C). Multigroup comparisons were performed by one-way ANOVA or two-way ANOVA if two conditions exist. Differences between the two groups were analyzed by both Student's t-tests. Statistical analysis was performed using the Prism graph pad and the difference was significant when P ⁇ 0.05.
  • Ectopic lipid accumulation is significantly upregulated in clear cell Renal Cancer Carcinmoa (Rezende et al., 1999, Differential diagnosis between monomorphic clear cell adenocarcinoma of salivary glands and renal (clear) cell carcinoma.Am J Surg Pathol) 23 , 1532-1538; Valera and Merino, 2011, Misdiagnosis of clear cell renal cell carcinoma.Nat Rev Urol 8 , 321-333).
  • RNF20 is known to act as a negative regulator of angiogenesis by inhibiting SREBP1c (Lee et al., 2014, Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.Hepatology 60 , 844- 857)
  • SREBP1c Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.Hepatology 60 , 844- 857)
  • the inventors investigated whether RNF20 is dysregulated in ccRCC tumors. As shown in FIG. 1A, RNF20 mRNA expression is significantly downregulated in ccRCC tumors compared to normal kidney tissue from the same patient.
  • RNA-Seq data from the Cancer Genome Atlas showed a significant decrease in RNF20 mRNA expression in ccRCC tumors, indicating that low RNF20 expression is closely associated with advanced tumor stages ( 1C).
  • immunohistochemistry (IHC) analysis showed that RNF20 protein expression was lower in ccRCC tumors than adjacent normal kidney tissues (FIG. 1D).
  • RNF20 staining data obtained from normal kidney and tumor tissues of the same patient also showed reduced RNF20 expression in ccRCC (FIG. 1E).
  • RNF20 expression was also reduced in ccRCC cell lines A498, Caki-2, and ACHN as compared to human primary renal cortical epithelial (HRCE) and HEK293 normal kidney cells (FIG. 9A). As shown in FIG. 16, RNF20 was also reduced in liver cancer cells at the protein and gene levels.
  • siRNA-mediated inhibition of RNF20 increased cell growth in ccRCC cell lines and liver cancer cell lines, including ACHN and A498 cells (FIGS. 1K and 9D, FIG. 20).
  • RNF20 was not overexpressed or siRNA-mediated knocked down, the levels of RNF20 did not affect the growth of high HRCE and HEK293 normal kidney cells (FIG. 9E-H).
  • the data means that RNF20 can act as a tumor suppressor in ccRCC cells independently of VHL status.
  • SREBP1 and adipose biosynthesis genes are expressed at high levels, which is also positively correlated with malignant progression and bad outcomes (Guo et al., 2009, EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.Sci Signal 2, ra82; Huang et al., 2012, Activation of androgen receptor, lipogenesis, and oxidative stress converged by SREBP -1 is responsible for regulating growth and progression of prostate cancer cells.Mol Cancer Res 10 , 133-142; Sun et al., 2015, SREBP1 regulates tumorigenesis and prognosis of pancreatic cancer through targeting lipid metabolism.Tumor Biol 36 , 4133-4141 ).
  • SREBP1 and lipophilic genes are associated with ectopic lipid storage in ccRCC. Therefore, the present inventors analyzed the expression pattern of the adipose biosynthetic gene in normal kidney and ccRCC tumor tissue. As shown in FIG. 2A, SREBP1c mRNA was significantly upregulated in ccRCC tumors compared to normal samples of the same patient (patient-matched). In addition, the TCGA RNA-Seq data indicated that SREBP1 was upregulated in ccRCC tumors (FIG. 2B) and had a positive correlation with advanced tumor stages (FIG. 2C).
  • mRNA levels of SREBP1 target genes for FASN and SCD1 were elevated in ccRCC tumors (FIGS. 10A-F).
  • the protein expression of SREBP1 and the lipophilic enzymes FASN and SCD1 increased simultaneously in ccRCC tumors compared to normal kidney tissue of the same patient, while RNF20 protein was downregulated (FIG. 2D).
  • mRNA expression of RNF20 is inversely correlated with that of the SREBP1c target genes FASN and ELOVL6 (FIGS. 10G and H), and FASN mRNA expression is positively associated with low survival (FIG. 2G).
  • mRNA levels of SREBP2 and its target gene, HMGCR, a gene that limits the rate of cholesterol synthesis were reduced in ccRCC tumors in qRT-PCR and TCGA RNASeq assays (FIG. 10 I-L).
  • intracellular triglyceride accumulation was greater in SREBP1c-overexpressing ACHN cells than in control ACHN cells (FIG. 3C).
  • inhibition of RNF20 increased intracellular triglyceride levels (FIG. 11D).
  • Inhibition of RNF20 using siRNF20 as described above was found to increase the SREBP1 protein concentration and increase the fat biosynthesis in liver cancer cell lines (FIGS. 21 and 22).
  • SREBP1c overexpression promoted mRNA expression of cell cycle regulators including PCNA, cyclin A, D1, and E in ccRCC cells (FIG. 3D), and further, RNF20 overexpression reduced the effect of overexpressed SREBP1c (FIG. 3D).
  • RNF20 knockdown also promoted cell cycle gene expression in ccRCC cells (FIG. 11E).
  • RNF20 overexpression reduced colony formation whereas ectopic SREBP1c expression increased colony formation in ACHN cells (FIG. 3E).
  • SREBP1c overexpression enhanced ccRCC cell proliferation (FIG. 3F) and knockdown of SREBP1 decreased ccRCC cell proliferation in both control and RNF20-inhibited cells (FIG. 11F). The same results were observed in liver cancer cell lines, and the results are described in FIGS. 17, 18 and 19.
  • RNF20 inhibits ccRCC cell proliferation by inhibiting SREBP1c-induced fat biosynthesis and cell cycle progression, indicating that RNF20 can be usefully used as a therapeutic agent for kidney cancer.
  • SREBP inhibitor betulin inhibits ccRCC cell proliferation by inhibiting SREBP1 and adipose biosynthesis irrespective of the VHL gene mutation (FIGS. 5A-C).
  • betulin eliminated cell proliferation and increased lipogenic activity after inhibition of RNF20 (FIGS. 13A-D).
  • SREBP1 and lipogenesis were overactivated with RNF20 downregulation in ccRCC (FIGS. 1 and 2), free SREBP1 was shown to promote ccRCC tumor development through activating lipogenesis.
  • SREBP1 and adipose biosynthesis pathways can be therapeutic targets for ccRCC.
  • SREBP1c As transcriptional activators, SREBP1c has been reported to stimulate fatty acid metabolism and cell cycle progression (Bengoechea-Alonso and Ericsson, 2006, Cdk1 / cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis. Cell Cycle 5 , 1708-1718; Jeon et. al., 2013, An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis.Cell Metab 18, 51-61; Williams et al., 2013, An essential requirement for the SCAP / SREBP signaling axis to protect cancer cells from lipotoxicity.Cancer Res 73 , 2850-2862).
  • PTTG1 was identified as a new target gene of SREBP1c (FIG. 4A).
  • PTTG1 expression was significantly reduced in kidney, liver and adipose tissue of SREBP1c deficient mice compared to wild type mice.
  • SREBP1c-overexpressing ACHN cells we examined PTTG1 expression in SREBP1c-overexpressing ACHN cells and found that ectopic SREBP1c expression increased PTTG1 mRNA expression, while RNF20 co-expression attenuated this effect (FIG. 4B).
  • RNF20 did not inhibit PTTG1 mRNA (FIG. 4C) or protein (FIG. 4D) expression.
  • SREBP1c promoted PTTG1 protein expression in ccRCC cells (FIG. 4E, lane 3).
  • siRNA mediated inhibition of PTTG1 did not affect SREBP1c or FASN protein expression (FIG. 4E, lane 4).
  • SREBP1c directly modulates PTTG1 transcription
  • the inventors have identified an EBOX motif and a potential SER (binding regulatory site), a binding site for SREBP1c at the proximal promoter site of PTTG1 gene in humans, monkeys, dogs, mice and rats. element) was analyzed (FIG. 12B).
  • PTTG1 is also involved in cell cycle progression and tumorigenesis in ccRCC, acting as a novel target gene for SREBP1c. It was also found that high expression of PTTG1 is closely associated with advanced tumor stage and low survival in ccRCC patients (FIGS. 4I and K). In addition, SREBP1 strongly stimulated mRNA and protein expression of PPTTG1, and several cell-cycle regulators, resulting in cancer cell proliferation in ccRCC (FIGS. 3 and 4). In contrast, RNF20 overexpression inhibited PTTG1 in both ccRCC cells and xenograft tumors (FIG. 4B; FIGS. 7D and E), and mRNA and protein levels of PTTG1 were increased by RNF20 inhibition (FIGS. 4C and D).
  • Betulin is a pharmacological inhibitor that exhibits a lipid-lowering effect by inhibiting the proteolytic process of SREBP protein (Soyal et al., 2015, Targeting SREBPs for treatment of the metabolic syndrome.Trends Pharmacol Sci 36 , 406-416; Tang et al., 2011, Inhibition of SREBP by a small molecule, betulin, improves hyperlipidemia and insulin resistance and reduces atherosclerotic plaques.Cell Metab 13 , 44-56). Betulin also attenuates the growth of various cancers by inhibiting various tumor factors, including cell-cycle regulators (Chintharlapalli et al., 2007, Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors.
  • Betulin inhibits lung carcinoma proliferation through activation of AMPK signaling.Tumor Biol 35 , 11153-11158).
  • the present inventors investigated the effect of betulin on SREBP inhibition to determine the anti-tumor effect on ccRCC cells.
  • Betulin treatment of VHL wild-type ACHN and VHL-depleted A498 ccRCC cells resulted in a decrease in nuclear SREBP1 protein in a dose-dependent manner, whereas the precursor morphology of SREBP1 was not affected (FIG. 5A). Means to suppress processing.
  • protein levels of cell cycle regulators including PTTG1 and cyclins B1 and E, were reduced in betulin-treated ccRCC cells (FIG. 5A).
  • RNF20 can inhibit the growth of ccRCC cells through mediating SREBP1c control and regulating lipobiosynthesis and cell cycle regulatory gene expression.
  • PTTG1 inhibition did not alter the mRNA expression of SREBP1c or FASN (FIG. 6B), whereas ectopic SREBP1c expression did not affect cell-cycle regulators including PCNA, cyclin A, D1, and E in ccRCC cells and mRNA of PTTG1. Expression was promoted (FIGS. 6B and C). In contrast, inhibition of PTTG1 expression in ccRCC cells downregulated these cell-cycle proteins (FIG. 6C). In addition, inhibition of PTTG1 inhibited cell proliferation in both control and SREBP1c-overexpressing ACHN cells (FIG. 6D).
  • FIG. 6A we measured PTTG1 expression and cell proliferation in the presence or absence of the ACC inhibitor TOFA or FASN inhibitor C75 (FIG. 6A).
  • TOFA and C75 as shown in FIG. 6E reduced intracellular triglyceride accumulation in ACHN cells.
  • lipophilic activity by TOFA or C75 did not significantly affect the mRNA expression of PTTG1 or cell-cycle regulator PCNA, Cyclin A, D1 and E (FIG. 6F; FIGS. 14A and B).
  • siRNA-mediated inhibition of FASN did not significantly alter the mRNA levels of PTTG1 and cell-cycle regulatory genes (FIGS. 14D and E).
  • ccRCC cell proliferation was significantly reduced in both cases where FASN was C75-mediated pharmacologically inhibited or siRNA-induced FASN knocked down (FIGS. 14C and F). Furthermore, the pharmacogenetic inhibition of FASN reduced the effect of SREBP1c overexpression on cell growth in ACHN cells, meaning that adipose biosynthesis inhibition could attenuate ccRCC cell proliferation via the SREBP1c-dependent pathway ( 6G and F).
  • PTTG1 expression is inversely correlated with RNF20 expression, reflecting the regulation of SREBP1c in ccRCC tumor tissue (FIGS. 4J and 8) and RNF20 downregulation, in part, upwards PTTG1 Regulation promotes ccRCC development and progression.
  • siRNA knockdown of PTTG1 resulted in decreased mRNA expression of cell cycle regulatory genes without altering adibiosynthetic activity (FIGS. 6C and E).
  • PTTG1 inhibition weakened the effect of activated SREBP1c on ccRCC cell proliferation (FIG. 6D).
  • RNF20-SREBP1c-PTTG1 axis is the center of ccRCC cell proliferation and tumorigenesis.
  • SREBP1 regulates fat biosynthesis by significantly increasing new fat biosynthesis. Therefore, the present inventors tested whether liposynthesis is associated with PTTG1 expression in the presence of novel lipophilic genetic pharmacological inhibitors, and the mRNA expression of PTTG1 in ACHN ccRCC cells was evaluated by pharmacological inhibition of lipobiosynthesis using TOFA or C75. 6F) or siRNA mediated knockdown of FASN (FIG. 14D). This means that PTTG1 is induced independent of the mechanism of lipophilic activation by SREBP1c. Thus, SREBP1c can influence different lipogenesis and cell cycle progression by regulating different target gene families, ultimately promoting tumor development in ccRCC.
  • RNF20 attenuated mRNA expression of SREBP1c, cell-cycle regulators, and lipophilic genes in xenograft tumors (FIG. 7E).
  • H & E staining ACHN tumors with high RNF20 expression showed a reduced number of cells with clear cell morphology (FIG. 7F).
  • oil red O staining lipid accumulation was reduced by ectopic RNF20 expression (FIG. 7F).
  • cell proliferation was reduced in xenograft tumors by exogenous RNF20 expression in Ki67 staining assay (FIG. 7F).
  • apoptosis was induced in xenograft tumors by RNF20 overexpression in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (FIG. 7F).
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • mRNA expression of pro-apoptotic genes including Bax, Bid, and caspase-3, is increased after ectopic RNF20 expression, whereas anti-apoptotic genes Bcl-2, cIAP-2, and XIAP -MRNA expression of apoptotic genes was observed to decrease (Fig. 15).
  • results indicate that overexpression of RNF20 can inhibit the growth of cancer by inhibiting SREBP1c expression and adipose biosynthesis (FIG. 7).
  • results also indicate that redirection of RNF20 in ccRCC leads to increased expression of SREBP1c, which in turn leads to increased expression of PTTG1 and lipophilic genes in ccRCC (FIG. 8), leading to tumor growth and progression.
  • RNF20 acts as a tumor suppressor by inhibiting SREBP1c-mediated adipose biosynthesis and cell cycle regulation (FIG. 8).
  • the data indicate that RNF20 downregulation promotes tumorigenesis by activating SREBP1c in ccRCC tumors, and down expression of RNF20 may act as a marker of cancer.
  • SREBP1c induced a PTTG1 in ccRCC to identify a new mechanism for stimulating cell cycle progression, and RNF20 can regulate the SREBP1c-lipid biosynthesis axis and the SREBP1c-PTTG1 axis.
  • the present invention demonstrates that the new therapeutic approaches that target RNF20-SREBP1c-lipid biosynthesis axis and RNF20-SREBP1c-PTTG1 axis pathways in cancer and RNF20 are novel tumor suppressors in ccRCC. It is present.

Abstract

Disclosed in the present application is a use of RNF20 as a therapeutic agent for kidney or liver cancer and as a marker for diagnosing kidney or liver cancer. In addition, disclosed in the present application is a method for screening for therapeutic agents for cancer associated with an abnormality of RNF20 by using a novel molecular mechanism, on the basis of RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipogenesis pathway.

Description

RNF20의 신장암 또는 간암 진단, 치료 및 치료제 스크리닝 용도Screening of RNF20 for the diagnosis, treatment and treatment of kidney or liver cancer
본원은 RNF20의 이상과 관련된 암의 진단, 치료 및 치료 약물 개발과 관련된 기술이다.The present application is a technique related to the diagnosis, treatment and treatment drug development of cancer associated with abnormalities of RNF20.
신장암은 60~70대의 노년층에서 주로 발생하고 지속적인 증가 추세를 보이고 있으며, 우리나라에서도 남성에서 발생하는 암 중 2.0%로 10위, 여성에서는 1.2%로 15위를 차지하고 있다. 신세포암에는 투명세포형 신세포암(clear cell RCC), 유두형 신세포암(papillary RCC), 혐색소형 신세포암(chromophobe RCC), 수질형 신세포암(medullary RCC), 분류불능 신세포암(unclassified RCC), 신이행상피암(kidney transitional cell carcinoma, TCC), 신장 호산성과립세포종(ranal oncocytoma) 등이 있다. 이 중 투명세포형 신세포함이 66~75%를 차지하고, 유두형 신세포암이 약 15%를 차지하며, 혐색소형 신세포암이 약 5%를 차지한다.Kidney cancer occurs mainly in elderly people in their 60's and 70's, and continues to increase, and Korea ranks 10th with 2.0% of cancers occurring in men and 15th with 1.2% in women. Renal cell carcinoma includes clear cell RCC, papillary RCC, chromophobe RCC, medullary RCC, and nonclassifiable renal cell carcinoma. (unclassified RCC), kidney transitional cell carcinoma (TCC), and renal eosinophilic granuloma (ranal oncocytoma). Clear cell type renal cell carcinoma accounted for 66-75%, papillary renal cell carcinoma accounted for 15%, and anachromosomal renal cell carcinoma accounted for 5%.
신장암은 종양의 크기가 작을 때는 증상이 거의 없으며, 종양이 어느 정도 커져서 장기를 밀어낼 정도가 되어야 비로소 증상이 나타난다. 따라서 진단이 늦어지는 경우가 많아 처음 진단될 때 환자의 30% 정도는 이미 전이된 상태로 나타나게 되므로, 초기에 신장암을 진단할 수 있는 방법이 필요하다. Kidney cancer has little symptoms when the tumor is small, and symptoms do not appear until the tumor is large enough to push the organs. Therefore, since the diagnosis is often delayed, about 30% of the patients are already metastasized when the first diagnosis is made. Therefore, a method for early diagnosis of kidney cancer is needed.
현재 신장암 진단에 사용되고 있는 방법은 복부초음파, 복부 전산화단층촬영(CT) 등이 이용되는데, 고가의 장비를 사용하므로 스크리닝의 목적으로 사용하기에는 경제성이 떨어지는 단점이 있다.Currently, methods used for diagnosing kidney cancer include abdominal ultrasonography, abdominal computed tomography (CT), etc., and use of expensive equipment has a disadvantage in that it is inexpensive to use for screening purposes.
또한 간세포암(Hepatocellular carcinoma, HCC)은 성인 간암에서 가장 흔한 유형으로, 암으로 인한 사망원인 중 세 번째를 차지한다(Stefaniuk P, et al., 2010, World J Gastroenterol 16: 418-424). HCC는 상당히 진행 되서야 증상이 나타나는 질환으로 이로 인해 적절한 치료시기를 놓치는 경우가 빈번하고, 치료를 하는 경우도 예후가 극히 나쁘다. 특히 수술로 절제가 불가능한 경우는 1년 내 사망하는 심각한 질환으로, 진단방법이 개선이 될 경우, 치료에 있어서는 상당한 개선이 기대된다. Hepatocellular carcinoma (HCC) is also the most common type of adult liver cancer and is the third leading cause of death from cancer (Stefaniuk P, et al., 2010, World J Gastroenterol 16: 418-424). HCC is a disease that manifests only when it progresses considerably, which often leads to a missed time for proper treatment and a very poor prognosis. In particular, surgical removal of the disease is a serious disease that dies within one year. When the diagnostic method is improved, a significant improvement in treatment is expected.
RNF20(Ring finger protein 20, RNF20)은 전사조절, DNA 손상 반응, 줄기세포 분화 및 지방생합성에 다양한 역할을 하는 E3 유비퀴틴 리가아제이다. RNF20은, 유전자 서브세트의 전사를 조절하고 크로마틴 리모델링에 기여하는 히스톤 H2B의 모노유비퀴틴화를 촉진시킨다(Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10, 483-488 ; 상기 Shema et al., 2011). Ring finger protein 20 (RNF20) is an E3 ubiquitin ligase that plays a variety of roles in transcriptional regulation, DNA damage response, stem cell differentiation and adipose synthesis. RNF20 promotes monoubiquitination of histone H2B, which regulates transcription of gene subsets and contributes to chromatin remodeling (Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10 , 483-488; Shema et al., 2011).
미국 공개특허공보 제2011-0081362호는 암치료에 관한 것으로, RAS 활성 돌연변이를 포함하는 암세포의 성장 또는 생존을 선택적으로 억제하기 위해 RNF20을 포함하는 다수의 세포내 과정에 관여하는 조절자를 억제하는 기술이 개시되어 있다. US Patent Publication No. 2011-0081362 relates to the treatment of cancer, a technique for inhibiting modulators involved in a number of intracellular processes including RNF20 to selectively inhibit the growth or survival of cancer cells comprising RAS active mutations. Is disclosed.
미국 공개특허공보 제2013-0295584호는 히스톤 단백질 폴리유비퀴톤화를 암 바이오마커의 용도에 관한 것으로, 히스톤 2B의 모노폴리퀴틴화를 갑상선암의 동정에 사용하며, CDC73 및 RNF20 사이의 상호작용에 근거한 히스톤 단백질의 모노유비퀴틴화를 조절하는 물질을 스크리닝하는 방법을 개시한다. US Patent Publication No. 2013-0295584 relates to the use of histone protein polyubiquitonation in cancer biomarkers, which uses monopolyquitinization of histone 2B in the identification of thyroid cancer, based on the interaction between CDC73 and RNF20. Disclosed are methods for screening substances that modulate monoubiquitination of histone proteins.
그러나 RNF20이 감소 또는 결실된 암 및 이의 발현 증가를 통한 암의 진단 또는 암치료제로서의 용도 및 그 기전은 알려져 있지 않다.However, the use and mechanisms of cancer as a diagnostic or cancer treatment of cancer through decreased or deleted RNF20 and increased expression thereof are unknown.
본원은 새로운 분자 기전에 근거한 RNF20의 이상과 관련된 암 치료제, 진단 마커 및 치료제 스크리닝 방법을 제공하고자 한다.The present application seeks to provide cancer therapeutic agents, diagnostic markers and therapeutic screening methods associated with abnormalities of RNF20 based on novel molecular mechanisms.
한 양태에서 본원은 RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현을 증가시키는 물질을 포함하는 RNF20의 감소 또는 결실과 관련된 암, 특히 고형암, 특히 신장암 또는 간암의 예방 또는 치료용 약학 조성물을 제공한다. RNF20은 신장암 및 간암에서 그 발현이 현저히 감소되어 있으며, 인비보 및 인비트로에서 상기 유전자 또는 단백질의 발현 또는 내인성 RNF20 유전자의 탈메틸화 또는 과메틸화 억제를 통한 과발현시 종양이 억제되었다. In one embodiment the present application relates to cancer associated with a reduction or deletion of RNF20, including a ring finger protein 20 (RNF20) gene, or a protein encoded by said gene, or a substance that increases expression of said gene, in particular solid cancer, in particular kidney cancer Or it provides a pharmaceutical composition for the prevention or treatment of liver cancer. The expression of RNF20 is markedly reduced in renal and liver cancers, and tumors are suppressed upon overexpression through inhibition of demethylation or hypermethylation of the gene or protein in vivo and in vitro, or by demethylation of the endogenous RNF20 gene.
일 구현예에서 본원에 따른 조성물이 사용되는 신장암은 특히 VHL 유전자 변이가 원인이 아닌, 신장암을 포함한다. In one embodiment kidney cancer in which the composition according to the invention is used comprises kidney cancer, in particular not caused by VHL gene mutations.
다른 양태에서 본원은 또한 RNF20(Ring finger protein 20) 검출용 물질을 포함하는, RNF20의 감소 또는 결실과 관련된 암, 특히 고형암, 특히 신장암 또는 간암의 진단 또는 예후 예측용 조성물을 제공한다. 암 조직 또는 세포에서 RNF20는 유전자 또는 단백질 수준에서 측정될 수 있고, 이를 근거로 신장암 또는 간암의 검출, 판단, 진단 또는 생존 예후 예측이 가능하다. In another aspect, the present invention also provides a composition for the diagnosis or prognosis of cancer, in particular solid cancer, in particular renal cancer or liver cancer, associated with a reduction or deletion of RNF20, comprising a substance for detecting ring finger protein 20 (RNF20). In cancer tissues or cells, RNF20 can be measured at the gene or protein level, and can be used to detect, judge, diagnose, or predict survival prognosis for kidney or liver cancer.
또 다른 양태에서 본원은 또한 RNF20-SREBP1c-PTTG1 신호전달 경로를 표적으로 하는 RNF20의 감소 또는 결실과 관련된 암, 특히 고형암, 더욱 특히, 신장암 또는 간암의 예방 또는 치료제 스크리닝 방법에 관한 것이다.In another aspect the present invention also relates to a method for screening or preventing or treating cancer, in particular solid cancer, more particularly renal or liver cancer, associated with a reduction or deletion of RNF20 targeting the RNF20-SREBP1c-PTTG1 signaling pathway.
일 구현예에서 상기 방법은 RNF20의 발현이 감소 또는 결여된 세포를 제공하는 제 1 단계; 상기 세포를 RNF20의 발현을 증가시킬 것으로 기대되는 시험물질로 처리하는 제 2 단계; 상기 시험물질로 처리된 세포에서 상기 SREBP1c 또는 PTTG1 중 하나 이상의 발현을 측정하는 제 3 단계; 및 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 SREBP1c 또는 PTTG1 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 SREBP1c 또는 PTTG1의 발현이 상기 대조군의 SREBP1c 또는 PTTG1 발현과 비교하여 감소한 경우 이를 RNF20의 발현 이상과 관련된 암의 치료제 후보물질로 선별하는 제 4 단계를 포함한다. In one embodiment the method comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase expression of RNF20; A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And comparing the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance and the control cells not in contact with the test substance, so that the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance is less than that of the control. And a fourth step of selecting it as a therapeutic agent candidate for cancer associated with abnormal expression of RNF20, if it has decreased in comparison with.
일 구현예에서 제 3 단계에서, 상기 SREBP1c에 발현을 측정하는 대신에, 또는 SREBP1c에 발현 측정에 부가하여, 상기 SREBP1에 의해 발현이 촉진되는 지질생합성에 관여하는 단백질 또는 그 유전자의 발현을 측정하는 단계를 포함할 수 있다. 이 경우 상기 제 4 단계에서 상기 시험물질과 접촉된 세포에서 상기 지질생합성에 관여하는 유전자 또는 단백질 발현은 상기 시험물질과 접촉되지 않은 대조군 세포와 비교하여 감소한 경우, 후보 물질로 선별한다. In one embodiment, in the third step, instead of measuring expression in the SREBP1c, or in addition to measuring expression in SREBP1c, measuring the expression of a protein or a gene involved in lipid biosynthesis in which expression is promoted by the SREBP1c is measured. It may include a step. In this case, if the expression of the gene or protein involved in the lipid biosynthesis in the cell contacted with the test substance in the fourth step is reduced compared to the control cell not contacted with the test substance, the candidate substance is selected.
다른 구현예에서 RNF20의 발현이 감소 또는 결여된 세포를 제공하는 제 1 단계; 상기 세포를 RNF20의 발현을 증가시키거나 또는 SREBP1c 발현을 억제시킬 것으로 기대되는 시험물질로 처리하는 제 2 단계; 상기 시험물질로 처리된 세포에서 PTTG1의 발현을 측정하는 제 3 단계; 및 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 PTTG1 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 PTTG1의 발현이 상기 대조군의 PTTG1 발현과 비교하여 감소한 경우 이를 신장암 치료제 후보물질로 선별하는 제 4 단계를 포함한다. In another embodiment, a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase the expression of RNF20 or inhibit SREBP1c expression; A third step of measuring expression of PTTG1 in cells treated with the test substance; And comparing the PTTG1 expression in the cells contacted with the test substance and the control cells not in contact with the test substance, and when the expression of the PTTG1 in the cell contacted with the test substance decreased compared with the PTTG1 expression of the control. And a fourth step of selecting a candidate for treating a kidney cancer.
또는 다른 양태에서 상기 제 3 단계 대신에, 상기 SREBP1c에 의해 발현이 촉진되는 지질생합성에 관여하는 단백질 또는 그 유전자의 발현을 측정하는 단계를 포함하며, 그리고 상기 제 4 단계 대신에, 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 지질생합성에 관여하는 유전자 또는 단백질 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 지질생합성에 관여하는 유전자 또는 단백질의 발현이 상기 대조군의 지질생합성을 촉진하는 유전자 또는 단백질의 발현과 비교하여 감소한 경우 이를 신장암 치료제 후보물질로 선별하는 단계를 포함한다. 본원에서는 신장암 또는 간암의 발생에 관여하는 두 종류의 독립적인 경로를 규명하였으며, 이는 특히 각각 기존에 신장암의 발병원인으로 알려진 VHL 기전과 독립적인 치료제 발굴에 사용될 수 있다. Or in another embodiment, instead of the third step, measuring the expression of a protein or gene thereof involved in lipid biosynthesis that is promoted by SREBP1c, and instead of the fourth step, Comparing the expression of the gene or protein involved in the lipid biosynthesis in the contacted cells and control cells not in contact with the test substance, the expression of the gene or protein involved in the lipid biosynthesis in the cell contacted with the test substance is If it is reduced compared to the expression of a gene or protein that promotes lipid biosynthesis, the step of selecting it as a candidate for the treatment of kidney cancer. We have identified two independent pathways involved in the development of renal or hepatic cancer, which can be used to find therapeutic agents that are independent of the VHL mechanism, which is known to be the cause of renal cancer.
또 다른 양태에서 본원은 신장암 또는 간암의 진단 또는 예후에 필요한 정보를 제공하기 위하여, 검사 대상체 유래의 생물학적 시료로부터 RNF20 바이오마커의 핵산 및/또는 단백질의 수준을 검출하는 단계; 상기 핵산 및/또는 단백질 수준 검출결과를 대조군 시료의 해당 마커의 상응하는 결과와 비교하는 단계; 및 상기 대조군 시료와 비교하여, 상기 대상체 유래 시료의 핵산 또는 단백질 수준에 변화가 있는 경우, 상기 대상체의 신장암 또는 간암의 진단 또는 생존 예후와 연관시키는 단계를 포함하는, RNF20 바이오마커를 검출하는 방법, 또는 신장암 또는 간암의 진단 또는 예후 판단 방법을 제공한다.In another aspect, the present invention provides a method for detecting a prognosis of a RNF20 biomarker from a biological sample from a test subject to provide information necessary for the diagnosis or prognosis of renal or liver cancer; Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And associating with a diagnosis or survival prognosis of kidney or liver cancer of the subject when there is a change in the nucleic acid or protein levels of the subject-derived sample compared to the control sample. Or a diagnosis or prognosis for renal or liver cancer.
또 다른 양태에서 본원은 RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현을 증가시키는 물질을 포함하는 인비트로에서 신장암 세포의 RNF20-SREBP1c-PTTG1 신호전달 경로 조절용 용도 또는 키트를 제공한다. In another embodiment, the present disclosure provides RNF20-SREBP1c-PTTG1 signaling in kidney cancer cells in vitro, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene. Provided are uses or kits for pathway control.
또 다른 양태에서 본원은 RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현을 증가시키는 물질을 포함하는 인비트로에서 신장암 세포의 RNF20-SREBP1c-지질생합성 경로 조절용 용도 또는 키트를 제공한다. In another embodiment, the present application provides a RNF20-SREBP1c-lipid biosynthesis pathway of renal cancer cells in vitro, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene. Provides an adjustment use or kit.
본원에 따른 키트는 다양한 목적을 위해 인비트로에서 RNF20-SREBP1c-PTTG1, 또는 RNF20-SREBP1c-지질생합성 신호전달을 조절하는데 사용될 수 있다. Kits according to the invention can be used to modulate RNF20-SREBP1c-PTTG1, or RNF20-SREBP1c-lipid biosynthesis signaling in vitro for various purposes.
본원에 따른 RNF20은 신장암의 치료제 및 진단용 마커로서 유용하게 사용될 수 있으며 또한 본원에서 규명된 새로운 분자기전인 RNF20-SREBP1c-PTTG1 및 RNF20-SREBP1c-지방생합성 경로는 신장암 치료제 발굴에 유용하게 사용될 수 있다.RNF20 according to the present application can be usefully used as a therapeutic agent and diagnostic marker for kidney cancer, and the new molecular mechanisms RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipid biosynthesis pathways identified herein can be usefully used to find a renal cancer therapeutic agent. have.
도 1은 RNF20의 발현량이 신장암 환자의 종양 조직에서 감소된 것을 나타낸 것이다. (A) 동일 환자의 종양 및 정상 신장 조직에서 qRT-PCR 분석을 수행한 결과 종양 조직에서 RNF20의 mRNA 발현량이 감소되어 있다. RNF20 발현양은 동일 신장암 환자의 정상 신장 조직으로 보정하였다. (B) 신장암 환자의 종양 및 정상 신장 조직에서 TCGA RNA-Seq 데이터베이스 분석을 한 결과 신장암 환자의 종양 조직에서 RNF20의 발현량이 감소되어 있다. (C) 신장암 환자의 T stage에 따른 RNF20 발현량을 분석한 것이다. RNA-Seq 데이터베이스는 TCGA에서 얻었다. ##P < 0.01 정상 신장 조직과 비교; ###P < 0.001 정상 신장 조직과 비교. (D) 신장암 환자의 종양 조직 마이크로어레이에서 RNF20 항체에 대한 면역조직화학법 염색 사진을 나타낸 것이다. (E) 동일한 신장암 환자의 종양과 종양 근처 정상 신장 조직에 대한 면역조직화학법 염색 사진을 나타낸 것이다. RNF20가 검출된 대표적인 조직 사진이다. 기준자는 100μm을 나타낸다. (F) ACHN 신장암 세포주에 RG108 처리 유무에 따른(+; 250μM or ++; 1 mM) RNF20 mRNA 발현량 변화를 qRT-PCR을 통해 측정하였다. RNF20 mRNA 발현량은 Cyclophilin mRNA 발현량으로 보정하였다. RNF20 mRNA 발현량은 대조군에 대한 상대값으로 표현하였다. 자료는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. (G) TCGA 데이터베이스에 등록된 532명의 신장암 환자에 대한 카플란-마이어 생존곡선을 나타낸 것이다. 환자는 RNF20 mRNA 발현량 중위값에 따라 두 군으로 나누었으며, 두 군의 차이는 로그-랭크 검정으로 확인하였다. RNF20 발현량이 낮게 관찰되는 신장암 환자의 생존 예후가 불량하게 관찰되었다. (H와 I) VHL 돌연변이 상태에 따른 생존 곡선 분석 결과를 나타낸 것이다. (J) ACHN 및 A498 신장암 세포주에 GFP(Mock) 또는 RNF20 발현 아데노바이러스를 감염시킨 후 CCK-8 기법을 활용하여 세포증식률을 측정하였다. RNF20가 과발현에 의해 신장암 세포의 증식이 억제됨을 관찰하였다. 결과는 독립 표본 다섯 개의 평균 ± 표준편차를 나타낸다. CCK-8, 세포수 계산 키트(Cell Counting Kit-8). (K) ACHN 및 A498 신장암 세포주에 비특이적 대조 siRNA(siControl) 또는 RNF20 특이적 siRNA(siRNF20)를 처리한 후 세포성장 곡선을 CCK-8 기법으로 측정하였다. RNF20 억제에 의해 신장암 세포의 증식이 증가함을 관찰하였다. *P < 0.05, **P < 0.01.1 shows that the expression level of RNF20 is reduced in the tumor tissue of kidney cancer patients. (A) As a result of qRT-PCR analysis in tumors and normal kidney tissues of the same patient, mRNA expression level of RNF20 was reduced in tumor tissues. The expression level of RNF20 was corrected to normal kidney tissue of the same kidney cancer patient. (B) Analysis of TCGA RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed a decrease in the expression level of RNF20 in tumor tissues of kidney cancer patients. (C) Analysis of RNF20 expression according to the T stage of kidney cancer patients. RNA-Seq database was obtained from TCGA. ## P <0.01 compared to normal kidney tissue; ### P <0.001 Compared with normal kidney tissue. (D) Immunohistochemical staining of RNF20 antibody in tumor tissue microarray of kidney cancer patient is shown. (E) Immunohistochemical staining pictures of tumors and normal kidney tissue near the tumor of the same kidney cancer patient are shown. RNF20 is a representative tissue photograph detected. Criteria represent 100 μm. (F) The change in RNF20 mRNA expression according to the presence or absence of RG108 treatment in the ACHN kidney cancer cell line (+; 250 μM or ++; 1 mM) was measured by qRT-PCR. RNF20 mRNA expression level was corrected by Cyclophilin mRNA expression level. RNF20 mRNA expression was expressed relative to the control. The data represent the mean ± standard deviation of three independent samples. (G) Kaplan-Meier survival curves for 532 kidney cancer patients enrolled in the TCGA database. The patients were divided into two groups according to the median RNF20 mRNA expression, and the difference between the two groups was confirmed by log-rank test. Survival prognosis was observed in renal cancer patients with low levels of RNF20 expression. (H and I) Survival curve analysis results according to the VHL mutation status. (J) After the infection of ACHN and A498 kidney cancer cell lines with GFP (Mock) or RNF20 expressing adenovirus, the cell proliferation rate was measured using CCK-8 technique. It was observed that RNF20 inhibited the proliferation of renal cancer cells by overexpression. The results represent the mean ± standard deviation of five independent samples. CCK-8, Cell Counting Kit-8. (K) Cell growth curves were measured by CCK-8 technique after treatment of ACHN and A498 kidney cancer cell lines with nonspecific control siRNA (siControl) or RNF20 specific siRNA (siRNF20). Inhibition of RNF20 increased the proliferation of renal cancer cells. * P <0.05, ** P <0.01.
도 2는 신장암 환자 종양 조직에서 SREBP1 및 지방합성 유전자는 증가되어 있으며, 이는 RNF20 발현량과 역의 상관관계를 보인다는 것을 나타낸 것이다. (A) 동일 신장암 환자의 종양 및 정상 신장 조직에서 SREBP1c의 qRT-PCR 분석 결과 동일 환자의 정상 신장 조직에 비해 종양 조직에서 SREBP1c mRNA 발현량이 증가되어 있음을 관찰하였다. (B) 신장암 환자의 종양 및 정상 신장 조직에서 TCGA RNA-Seq 데이터베이스 분석을 한 결과 신장암 환자의 종양 조직에서 SREBP1의 발현량이 증가되어 있다. (C) 신장암 환자의 T stage에 따른 SREBP1 발현량 분석. RNA-Seq 데이터베이스는 TCGA에서 얻었다. ##P < 0.01 정상 신장과 비교; ###P < 0.001 정상 신장과 비교. (D) 동일 신장암 환자에서 종양과 정상 신장 조직의 단백질 양을 웨스턴 블랏팅 기법을 통해 분석하였다. 세포융해물을 SDS-PAGE에서 분리시킨 뒤 항체를 이용하여 웨스턴 블랏팅을 수행하였다. 단백질 양도 mRNA 발현량과 비슷한 패턴임을 관찰하였다. (E) 동일 신장암 환자에서 종양과 정상 신장 조직을 면역조직화학법으로 염색하였다. RNF20와 SREBP1 염색을 한 대표적인 조직 절편을 나타내었다. 기준자는 100μm을 나타내었다. (F) TCGA 데이터베이스에서 얻은 RNF20와 SREBP1 mRNA 발현량의 상관관계를 피어슨 상관 분석을 이용하여 계산한 결과 역의 상관관계를 나타내었다. 환자 수(n), 피어슨 상관계수(r), P 값(P). (G) FASN 발현량으로 나눈 군에서 분석한 카플란-마이어 생존 곡선. P 값은 로그-랭크 검정을 통해 계산하였다. FASN 발현량이 높게 관찰되는 신장암 환자의 생존 예후가 불량하게 관찰되었다.Figure 2 shows that SREBP1 and liposynthetic genes are increased in the tumor tissue of kidney cancer patients, which is inversely correlated with the expression level of RNF20. (A) As a result of qRT-PCR analysis of SREBP1c in tumors and normal kidney tissue of the same kidney cancer patient, it was observed that the amount of SREBP1c mRNA expression was increased in tumor tissues compared to normal kidney tissue of the same patient. (B) Analysis of TCGA RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed increased expression levels of SREBP1 in tumor tissues of kidney cancer patients. (C) Analysis of SREBP1 expression according to the T stage of kidney cancer patients. RNA-Seq database was obtained from TCGA. ## P <0.01 compared with normal height; ### P <0.001 Compared with normal height. (D) The protein levels of tumors and normal kidney tissues in the same kidney cancer patients were analyzed by Western blotting. Cell lysates were separated on SDS-PAGE and subjected to western blotting using antibodies. The protein amount was observed to be similar to the mRNA expression level. (E) Tumors and normal kidney tissues were stained by immunohistochemistry in the same kidney cancer patient. Representative tissue sections were shown with RNF20 and SREBP1 staining. Criteria showed 100 μm. (F) The correlation between the expression levels of RNF20 and SREBP1 mRNA obtained from the TCGA database was calculated using Pearson's correlation analysis. Number of patients (n), Pearson's correlation coefficient (r), P value (P). (G) Kaplan-Meier survival curves analyzed in the group divided by the amount of FASN expression. P values were calculated via log-rank test. The survival prognosis of renal cancer patients with high FASN expression was observed poorly.
도 3은 RNF20가 SREBP1을 억제하여 지방대사 생합성과 신장암 세포증식을 억제함을 나타낸 것이다. (A) ACHN 신장암 세포주에 Myc-RNF20 또는 Flag-SREBP1c을 발현하는 아데노바이러스를 감염시켰다. 세포융해물을 SDS-PAGE를 통해 분리한 후 항체를 이용하여 웨스턴 블랏팅을 수행하였다. RNF20와 SREBP1c의 과발현 검증. (B) ACHN 신장암 세포주에 렌티바이러스를 이용하여 RNF20 또는 SREBP1c를 지속적으로 과발현시켰다. 상대적인 mRNA 발현량은 qRT-PCR을 통해 측정하였다. 각 유전자의 mRNA 양은 GAPDH 유전자의 mRNA 발현량으로 보정하였다. 각 mRNA 발현량은 Mock 대조군에 대한 상대값으로 표현하였다. (C) 렌티바이러스를 통한 RNF20 또는 SREBP1c 과발현 세포 내 트리글리세리드 양을 측정하였다. 그 결과 RNF20 과발현에 의해 지방대사물 축적량이 감소하였다. (D) RNF20 또는 SREBP1c가 과발현된 ACHN 신장암 세포주에서 세포주기 조절유전자의 발현량을 qRT-PCR로 분석하였다. (E) RNF20 또는 SREBP1c가 과발현된 ACHN 신장암 세포주에서 콜로니 형성능력을 크리스탈 바이올렛 염색으로 측정하였다. (F) RNF20 또는 SREBP1c가 과발현된 ACHN 신장암 세포주에서 CCK-8 기법을 활용하여 세포증식률 곡선을 분석하였다. 결과는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. #P < 0.05 Mock 군과 비교; ##P < 0.01 Mock 군과 비교; ###P < 0.001 Mock 군과 비교; *P < 0.05; **P < 0.01; ***P < 0.001.Figure 3 shows that RNF20 inhibits SREBP1 inhibits the metabolic biosynthesis and renal cancer cell proliferation. (A) ACHN kidney cancer cell lines were infected with adenoviruses expressing Myc-RNF20 or Flag-SREBP1c. Cell lysates were separated via SDS-PAGE and Western blotting was performed using antibodies. Validation of overexpression of RNF20 and SREBP1c. (B) RNF20 or SREBP1c were continuously overexpressed using lentiviral in ACHN kidney cancer cell lines. Relative mRNA expression was measured by qRT-PCR. MRNA amount of each gene was corrected by mRNA expression amount of GAPDH gene. Each mRNA expression amount was expressed relative to the mock control. (C) The amount of triglycerides in RNF20 or SREBP1c overexpressing cells via lentivirus was measured. As a result, the amount of fat metabolites decreased due to overexpression of RNF20. (D) The expression levels of cell cycle regulator genes in ACHN kidney cancer cell lines overexpressing RNF20 or SREBP1c were analyzed by qRT-PCR. (E) Colony-forming ability in ACHN kidney cancer cell lines overexpressing RNF20 or SREBP1c was measured by crystal violet staining. (F) Cell growth rate curves were analyzed using CCK-8 technique in ACHN kidney cancer cell lines overexpressing RNF20 or SREBP1c. The results represent the mean ± standard deviation of three independent samples. # P <0.05 compared with Mock group; ## P <0.01 compared with Mock group; ### P <0.001 compared with Mock group; * P <0.05; ** P <0.01; *** P <0.001.
도 4는 SREBP1c의 새로운 표적 유전자로 PTTG1을 동정한 것을 나타낸 것이다. (A) 정상 생쥐와 SREBP1c가 결핍된 생쥐의 간에서 RNA-Seq을 통해 분석한 전사체양을 산점도로 표현하였다. (B) RNF20 또는 SREBP1c가 과발현된 ACHN 신장암 세포주에서 PTTG1 mRNA 발현량을 분석하였다. PTTG1 mRNA 발현량은 GAPDH로 보정하였으며 대조군에 대한 상대값으로 표현하였다. (C) ACHN 신장암 세포주에 siRNF20 또는 siSREBP1을 형질도입한 후 PTTG1 mRNA 양을 qRT-PCR을 통해 분석하였다. PTTG1 mRNA 발현량은 GAPDH로 보정하였으며, 모든 mRNA 발현량은 대조군에 대한 상대값으로 표현하였다. (D) ACHN 신장암 세포에 siControl과 siRNF20를 형질도입한 후 항체를 이용하여 웨스턴 블랏팅을 수행하였다. RNF20 억제에 의해 SREBP1과 PTTG1이 단백질 수준에서 증가함을 관찰하였다. nSREBP1, 핵 SREBP1. (E) ACHN 신장암 세포주에 SREBP1c 아데노바이러스를 감염시키고 PTTG1 siRNA를 형질도입하였다. 항체를 이용하여 웨스턴 블랏팅을 수행하였다. SREBP1c 과발현에 의해 PTTG1 단백질 양이 증가함을 관찰하였다. (F) HEK293 세포주에 PTTG1 프로모터가 클로닝된 루시퍼레이즈 리포터 플라스미드와 베타-갈라토시데이즈, RNF20, 또는 SREBP1c 발현 벡터를 함께 형질도입시켰다. 세포융해물을 루시퍼레이즈와 베타-갈라토시데이즈 기법으로 분석하였다. 자료는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. RLU, 상대적인 형광도 (G) 동일 신장암 환자의 종양 및 정상 신장 조직에서 PTTG1의 qRT-PCR 분석 결과 PTTG1 mRNA 발현량은 신장암 종양 조직에서 증가되어 있음을 관찰하였다. (H) 신장암 환자의 종양 및 정상 신장 조직에서 TCGA RNA-Seq 데이터베이스 분석을 한 결과 신장암 환자의 종양 조직에서 PTTG1의 발현량이 증가되어 있다. (I) 신장암 환자의 T stage에 따른 PTTG1 발현량 분석하였다. RNA-Seq 데이터베이스는 TCGA에서 얻었다. ###P < 0.001 정상 신장과 비교; ###P < 0.001 정상 신장과 비교. (J) TCGA 데이터베이스에서 얻은 RNF20와 PTTG1 mRNA 발현량의 상관관계를 피어슨 상관 분석을 이용하여 계산한 결과 역의 상관관계를 나타내었다. 환자 수(n), 피어슨 상관계수(r), P 값(P) (K) PTTG1 발현량으로 나눈 군에서 분석한 카플란-마이어 생존 곡선을 나타내었다. P 값은 로그-랭크 검정을 통해 계산하였다. PTTG1 발현량이 높게 관찰되는 신장암 환자의 생존 예후가 불량하게 관찰되었다.Figure 4 shows the identification of PTTG1 as a new target gene of SREBP1c. (A) Scatter plots of transcriptomes analyzed by RNA-Seq in livers of normal mice and mice lacking SREBP1c were expressed. (B) PTTG1 mRNA expression levels in ACHN kidney cancer cell lines overexpressing RNF20 or SREBP1c were analyzed. PTTG1 mRNA expression was corrected by GAPDH and expressed as relative to the control. (C) After transducing siRNF20 or siSREBP1 to ACHN kidney cancer cell lines, the amount of PTTG1 mRNA was analyzed by qRT-PCR. PTTG1 mRNA expression level was corrected by GAPDH, and all mRNA expression levels were expressed relative to the control group. (D) siControl and siRNF20 were transduced into ACHN kidney cancer cells, and Western blotting was performed using antibodies. Inhibition of RNF20 increased SREBP1 and PTTG1 at protein levels. nSREBP1, nuclear SREBP1. (E) ACHN kidney cancer cell lines were infected with SREBP1c adenovirus and transduced PTTG1 siRNA. Western blotting was performed using the antibody. It was observed that the amount of PTTG1 protein was increased by SREBP1c overexpression. (F) The HEK293 cell line was transduced with a luciferase reporter plasmid cloned with a PTTG1 promoter and a beta-galactosidase, RNF20, or SREBP1c expression vector. Cell lysates were analyzed by luciferase and beta-galactidase techniques. The data represent the mean ± standard deviation of three independent samples. RLU, relative fluorescence (G) The qRT-PCR analysis of PTTG1 in tumors and normal kidney tissues of the same kidney cancer patients showed that PTTG1 mRNA expression was increased in kidney cancer tumor tissues. (H) Analysis of TCGA RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed increased expression of PTTG1 in tumor tissues of kidney cancer patients. (I) PTTG1 expression level according to T stage of kidney cancer patients was analyzed. RNA-Seq database was obtained from TCGA. ### P <0.001 compared with normal height; ### P <0.001 Compared with normal height. (J) The correlation between RNF20 and PTTG1 mRNA expression in the TCGA database was calculated using Pearson's correlation analysis. Kaplan-Meier survival curves were analyzed in the group divided by the number of patients (n), Pearson's correlation coefficient (r), and P value (P) (K) PTTG1 expression. P values were calculated via log-rank test. The survival prognosis of renal cancer patients with high PTTG1 expression was observed poorly.
도 5는 SREBP 억제제인 베툴린이 신장암 세포증식을 억제한다는 것을 나타내었다. (A) ACHN 및 A498 신장암 세포주에 다양한 농도의 베툴린을 12시간 동안 처리한 후 세포융해물을 SDS-PAGE로 분리하여 웨스턴 블랏팅을 수행하였다. 베툴린에 의해 SREBP1, 지방대사 생합성, 세포주기 조절단백질의 양이 감소함을 관찰하였다. pSREBP1, 전구체 SREBP1; nSREBP1, 핵 SREBP1. (B와 C) ACHN 및 A498 신장암 세포주에 베툴린을 처리하고 세포증식률을 CCK-8 기법을 통해 측정하였다. 베툴린 처리에 의해 신장암 세포증식률이 억제되었다. 자료는 독립 표본 다섯 개의 평균 ± 표준편차를 나타낸다. (D) ACHN 신장암 세포주에 베툴린을 처리한 후 세포를 고정하여 보디피(BODIPY, 녹색)와 다피(DAPI, 청색)로 염색하였다. 베툴린 처리에 의해 신장암 세포 내 지방대사물 축적량이 감소됨을 관찰하였다. 사진은 공초점현미경을 통해 얻었다. 기준자는 10 μm를 나타낸다. (E) ACHN 신장암 세포주에 베툴린을 24시간 처리한 후 세포를 고정하여 프로피디움 아이오딘화합물로 염색하였다. DNA 양은 유동세포분석법으로 측정하였다. 각 세포주기의 백분율을 표현하였다. 베툴린 처리에 의해 신장암 세포의 세포주기가 G1 단계에 머무는 것을 관찰하였다.5 shows that betulin, an SREBP inhibitor, inhibits kidney cancer cell proliferation. (A) ACHN and A498 kidney cancer cell lines were treated with various concentrations of betulin for 12 hours, and then cell lysates were separated by SDS-PAGE to perform western blotting. It was observed that betulin reduced the amount of SREBP1, fat metabolism biosynthesis, and cell cycle regulatory proteins. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1. (B and C) ACHN and A498 kidney cancer cell lines were treated with betulin and cell proliferation was measured via CCK-8 technique. Betulin treatment inhibited renal cancer cell proliferation. The data represent the mean ± standard deviation of five independent samples. (D) After treatment with betulin in the ACHN kidney cancer cell line, the cells were fixed and stained with bodiphy (BODIPY, green) and dapi (DAPI, blue). It was observed that betulin treatment reduced the amount of fat metabolites in kidney cancer cells. Photos were taken via confocal microscope. Criteria represent 10 μm. (E) ACHN kidney cancer cell line was treated with betulin for 24 hours, and then cells were fixed and stained with propidium iodine compound. DNA amount was measured by flow cytometry. The percentage of each cell cycle is expressed. Betulin treatment observed that the cell cycle of kidney cancer cells stayed at the G1 stage.
도 6은 SREBP1c가 세포주기와 지방대사를 조절하여 신장암의 세포 성장에 영향을 미친다는 것을 나타낸 것이다. (A) siRNA를 통한 PTTG1 또는 FASN 억제 및 약제를 통한 지방생합성 유전자(FASN, ACC)의 억제 효과를 검증하기 위한 실험 계획 모식도를 나타낸 것이다. (B와 C) 렌티바이러스를 통한 SREBP1c 과발현 ACHN 신장암 세포주에 PTTG1 siRNA를 형질도입하였다. 48시간 후 지방생합성과 세포주기 조절유전자들의 mRNA 발현량을 qRT-PCR을 통해 측정하고 GAPDH mRNA 발현량으로 보정하였다. 자료는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. (D) (B와 C)에 서술한 세포의 상대적인 증식률을 CCK-8 기법으로 측정하였다. (E) ACHN 신장암 세포주에 ACC 억제제 토파(TOFA, 10μg/ml) 또는 FASN 억제제 C75(10 μg/ml)를 24시간 동안 처리한 후 세포 내 트리글리세리드 양을 측정하였다. 지방생합성 억제에 따른 지방대사물 감소 효과를 검증한 것이다. (F) ACHN 신장암 세포주에 토파(10μg/ml) 또는 C75(10μg/ml)를 24시간 동안 처리한 후 PTTG1 mRNA 발현량을 qRT-PCR로 측정하였다. mRNA 양은 대조군에 대한 상대값으로 표현하였다. PTTG1 mRNA 발현량은 지방생합성 제어에 의해 변하지 않았다. (G) SREBP1c를 지속적으로 과발현하는 ACHN 신장암 세포주에 C75를 처리한 후 세포증식 속도를 CCK-8 기법을 통해 측정하였다. 자료는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. *P < 0.05, **P < 0.01; CCK-8, 세포수 계산 키트-8.Figure 6 shows that SREBP1c affects cell growth in kidney cancer by regulating cell cycle and fat metabolism. (A) An experimental design schematic for verifying the inhibitory effect of PTTG1 or FASN inhibition through siRNA and the fat biosynthesis gene (FASN, ACC) through the drug is shown. (B and C) PTRE1 siRNA was transduced into SREBP1c overexpressing ACHN kidney cancer cell line via lentiviral. After 48 hours, mRNA expression of adipose biosynthesis and cell cycle regulators was measured by qRT-PCR and corrected by GAPDH mRNA expression. The data represent the mean ± standard deviation of three independent samples. (D) Relative proliferation of the cells described in (B and C) was measured by CCK-8 technique. (E) ACHN kidney cancer cell lines were treated with ACC inhibitor Topa (TOFA, 10 μg / ml) or FASN inhibitor C75 (10 μg / ml) for 24 hours and then the amount of triglycerides in the cells was measured. The effect of fat metabolism reduction by inhibiting fat biosynthesis was verified. (F) ACHN kidney cancer cell lines were treated with topa (10 μg / ml) or C75 (10 μg / ml) for 24 hours and then PTTG1 mRNA expression was measured by qRT-PCR. mRNA amount was expressed relative to the control. The amount of PTTG1 mRNA expression did not change by the control of fat biosynthesis. (G) Cell growth rate was measured by CCK-8 technique after treatment with C75 in ACHN renal cancer cell lines that continuously overexpress SREBP1c. The data represent the mean ± standard deviation of three independent samples. * P <0.05, ** P <0.01; CCK-8, Cell Counting Kit-8.
도 7은 RNF20 과발현이 이종 이식 생쥐의 종양 증식을 억제한다는 것을 나타낸 것이다. (A) BALC/c 누드 생쥐에 Mock 또는 RNF20 과발현 ACHN 신장암 세포를 피하로 주사하여 종양을 생성시켰다. 각 군은 다섯 마리의 생쥐에서 유래한 10개의 종양을 의미한다. 실험 종료 시 종양의 크기를 보여주는 대표적 사진은 개체 내 이종 이식 종양 성장에 RNF20 과발현이 끼치는 영향을 보여준다. 생체 내에서 RNF20 과발현에 의한 항암효과 검증. 기준자는 10mm를 나타낸다. (B) RNF20 과발현에 따른 ACHN 신장암 세포의 이종 이식 종양 부피를 35일에 걸쳐 측정하였다. 도표는 종양 부피를 평균 ± 표준오차를 나타낸다. (C) 실험 종료 시 종양 무게를 측정하고 표현하였다. (D) ACHN 신장암 이종 이식 종양 내 RNF20, nSREBP1, PTTG1 및 FASN 단백질 양을 웨스턴 블랏팅 기법을 통해 분석하였다. nSREBP1, 핵 SREBP1. (E) ACHN 신장암 이종 이식 종양에서 RNF20가 세포주기 및 지방생합성 유전자 발현량 조절에 미치는 영향을 qRT-PCR을 통해 분석하였다. 각 유전자의 mRNA 양은 GAPDH 유전자의 mRNA 발현량으로 보정하고 Mock 대조군에 대한 상대값으로 표현하였다. (F) 신장암 이종 이식 종양의 조직학적 분석. Mock 또는 RNF20을 과발현하는 ACHN 이종 이식 종양 절편을 헤마톡실린 에오신(H/E) 또는 Oil red O로 염색하고 대표 사진을 실었다. 이종 이식 종양 절편을 Ki67과 TUNEL로 염색하였다. TUNEL, 말단데옥시뉴클레오티드전달효소 dUTP 틈새 말단 표지. RNF20 과발현에 의해 종양증식 억제 및 지방대사물 축적 감소효과를 검증하였다. 기준자는 100μm를 의미한다.Figure 7 shows that RNF20 overexpression inhibits tumor proliferation in xenograft mice. (A) BALC / c nude mice were injected subcutaneously with Mock or RNF20 overexpressing ACHN kidney cancer cells to generate tumors. Each group represents 10 tumors from five mice. Representative photographs showing tumor size at the end of the experiment show the effect of RNF20 overexpression on xenograft tumor growth in an individual. Validation of anticancer effect by RNF20 overexpression in vivo. Criteria represent 10 mm. (B) Xenograft tumor volume of ACHN kidney cancer cells following RNF20 overexpression was measured over 35 days. The plot shows the mean volume plus standard error. (C) Tumor weights were measured and expressed at the end of the experiment. (D) The amount of RNF20, nSREBP1, PTTG1 and FASN proteins in ACHN kidney cancer xenograft tumors were analyzed by Western blotting technique. nSREBP1, nuclear SREBP1. (E) The effect of RNF20 on cell cycle and adipose biosynthesis gene expression in ACHN kidney cancer xenograft tumors was analyzed by qRT-PCR. The mRNA amount of each gene was corrected by the mRNA expression level of the GAPDH gene and expressed as a relative value to the mock control group. (F) Histological analysis of kidney cancer xenograft tumors. ACHN xenograft tumor sections overexpressing Mock or RNF20 were stained with hematoxylin eosin (H / E) or Oil red O and representative pictures were taken. Xenograft tumor sections were stained with Ki67 and TUNEL. TUNEL, terminal deoxynucleotide transferase dUTP niche terminal label. RNF20 overexpression demonstrated the effects of tumor growth inhibition and fat metabolism accumulation. Standard means 100 μm.
도 8은 신장암에서 RNF20의 SREBP1 의존적 지방생합성과 세포주기 제어를 통한 종양억제 기능을 제안하는 모델(요약모델)을 나타낸 것이다. 신장암 환자에서 RNF20의 발현량 저하와 SREBP1의 발현량 증가는 생존 예후 불량과 관련이 있다. RNF20 억제는 SREBP1의 활성화를 유도하여 신장암 종양발생을 유도한다. SREBP1은 새로운 표적 유전자인 PTTG1을 통해 세포주기를 조절한다. 또한 SREBP1 억제제인 베툴린은 지방생합성 제어와 G1세포주기 정지를 통해 신장암 세포증식을 억제한다. 종합하면 RNF20 발현 감소는 SREBP1 의존적 지방생합성과 세포증식 활성을 통해 신장암의 종양발생을 촉진한다. FIG. 8 shows a model (summary model) suggesting SREBP1-dependent adipose biosynthesis of RNF20 and renal tumor suppression function through cell cycle control in kidney cancer. Decreased expression of RNF20 and increased expression of SREBP1 in kidney cancer patients are associated with poor survival prognosis. RNF20 inhibition leads to the activation of SREBP1 leading to renal cancer oncogenesis. SREBP1 regulates the cell cycle through a new target gene, PTTG1. Betulin, a SREBP1 inhibitor, also inhibits renal cancer cell proliferation through adipose biosynthesis control and G1 cell cycle arrest. Taken together, reduced RNF20 expression promotes tumorigenicity of renal cancer through SREBP1-dependent adipose biosynthesis and cell proliferation activity.
도 9는 RNF20가 신장암 세포에서 종양억제 기능을 보이나, 정상 신장 세포의 세포증식에는 영향을 미치지 않는다는 것을 나타낸 것이다. (A) 사람 초계 신장 피질 표피(HRCE; human primary renal cortical epithelial) 세포주, HEK293와 같은 정상 신장 세포주와 ACHN, A498, Caki-2와 같은 신장암 세포주에서 RNF20 단백질 발현량을 웨스턴 블랏팅 기법을 통해 분석하였다. (B) HEK293 세포주에 RG108(+; 250μM or ++; 1 mM)을 48시간 동안 처리한 후 RNF20 mRNA 발현량을 qRT-PCR을 통해 측정하였다. RNF20 mRNA 발현량은 Cyclophilin mRNA 발현량으로 보정하고 대조군에 대한 상대값으로 표현하였다. 자료는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. n.s., 유의미하지 않음. (C) ACHN 및 A498 신장암 세포주에 GFP(Mock) 또는 Myc-RNF20 발현 아데노바이러스를 감염시켰다. 24시간 후 세포융해물을 SDS-PAGE로 분리시킨 뒤 항체를 이용하여 웨스턴 블랏팅을 수행하였다. (D) ACHN 신장암 세포주에 siControl과 siRNF20를 형질도입한 후 RNF20 발현량을 웨스턴 블랏팅 기법을 통해 측정하였다. (E) HRCE 및 HEK293 세포주에 Mock 또는 Myc-RNF20 발현 아데노바이러스를 감염시킨 후 세포융해물을 SDS-PAGE를 통해 분리한 후 항체를 이용하여 웨스턴 블랏팅을 수행하였다. (F) HRCE 및 HEK293 세포에 RNF20 발현 아데노바이러스를 감염시킨 후 CCK-8 기법을 활용하여 생장곡선을 측정하였다. 자료는 독립 표본 다섯 개의 평균 ± 표준편차를 나타낸다. n.s., 유의미하지 않음. (G) HRCE 및 HEK293 세포에 siControl과 siRNF20를 형질도입한 후 세포융해물을 웨스턴 블랏팅으로 분석하였다. (H) HRCE 및 HEK293 세포에 siRNF20를 형질도입한 후 상대적인 세포 성장속도를 CCK-8 기법을 통해 측정하였다. 9 shows that RNF20 shows tumor suppression function in kidney cancer cells, but does not affect cell proliferation of normal kidney cells. (A) Western blotting technique for the expression of RNF20 protein in human primary renal cortical epithelial (HRCE) cell lines, normal kidney cell lines such as HEK293, and kidney cancer cell lines such as ACHN, A498, and Caki-2. Analyzed. (B) RG108 (+; 250 μM or ++; 1 mM) was treated in HEK293 cell line for 48 hours, and RNF20 mRNA expression was measured by qRT-PCR. The expression level of RNF20 mRNA was corrected by the expression level of Cyclophilin mRNA and expressed as a relative value to the control group. The data represent the mean ± standard deviation of three independent samples. n.s., not significant. (C) ACHN and A498 kidney cancer cell lines were infected with GFP (Mock) or Myc-RNF20 expressing adenovirus. After 24 hours, the cell lysate was separated by SDS-PAGE, followed by Western blotting using an antibody. (D) After transducing siControl and siRNF20 into ACHN kidney cancer cell lines, RNF20 expression was measured by Western blotting. (E) HRCE and HEK293 cell lines were infected with Mock or Myc-RNF20 expressing adenovirus and cell lysates were isolated via SDS-PAGE, followed by Western blotting using antibodies. (F) After HRN and HEK293 cells were infected with RNF20 expressing adenovirus, growth curves were measured using CCK-8 technique. The data represent the mean ± standard deviation of five independent samples. n.s., not significant. (G) After transducing siControl and siRNF20 to HRCE and HEK293 cells, cell lysates were analyzed by Western blotting. (H) After transducing siRNF20 to HRCE and HEK293 cells, the relative cell growth rate was measured by CCK-8 technique.
도 10은 신장암 종양 조직에서 지방생합성 효소의 발현이 증가되어 있으며 RNF20 발현과는 역의 상관관계를 보인다는 것을 나타낸 것이다. (A) 동일 신장암 환자의 종양 및 정상 신장 조직에서 FASN의 qRT-PCR 분석. mRNA 발현량은 동일 환자의 정상 신장 조직으로 보정하였다. (B) 신장암 환자의 종양 및 정상 신장 조직에서 보정된 FASN RNA-Seq 발현량을 나타낸 것이다. (C) 신장암 T stage에 따른 FASN 발현량을 분석한 것이다. (D) 동일 신장암 환자의 종양 및 정상 신장 조직에서 SCD1의 qRT-PCR 분석. mRNA 발현량은 동일 환자의 정상 신장 조직으로 보정하였다. (E) 신장암 종양 및 정상 신장 조직에서 보정된 SCD1 RNA-seq 발현량을 나타낸 것이다. (F) 신장암 T stage에 따른 SCD1 발현량을 분석한 것이다. ###P < 0.001 정상 신장 조직과 비교하였다. (G) TCGA 데이터베이스에서 얻은 RNF20와 FASN mRNA 발현량의 상관관계를 피어슨 상관 분석을 이용하여 계산하였다(역의 상관관계). (H) TCGA 데이터베이스에서 얻은 RNF20와 ELOVL6 mRNA 발현량의 상관관계를 피어슨 상관 분석을 이용하여 계산하였다(역의 상관관계). (I) 동일 신장암 환자의 종양 및 정상 신장 조직에서 SREBP2의 qRT-PCR 분석. mRNA 발현량은 동일 환자의 정상 신장 조직으로 보정하였다. (J) 신장암 종양 및 정상 신장 조직에서 보정된 SREBP2 RNA-seq 발현량을 나타낸 것이다. (K) 동일 신장암 환자의 종양 및 정상 신장 조직에서 HMGCR의 qRT-PCR 분석. mRNA 발현량은 동일 환자의 정상 신장 조직으로 보정하였다. (L) 신장암 종양 및 정상 신장 조직에서 보정된 HMGCR RNA-seq 발현량을 나타내었다. *P < 0.05; ***P < 0.001.Figure 10 shows that the expression of liposynthetic enzymes in renal cancer tumor tissue is increased and inversely correlated with RNF20 expression. (A) qRT-PCR analysis of FASN in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient. (B) shows the corrected FASN RNA-Seq expression in tumors and normal kidney tissue of kidney cancer patients. (C) Analysis of FASN expression according to renal cancer T stage. (D) qRT-PCR analysis of SCD1 in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient. (E) Corrected SCD1 RNA-seq expression levels are shown in renal cancer tumors and normal kidney tissues. (F) Analysis of SCD1 expression according to renal cancer T stage. ### P <0.001 compared with normal kidney tissue. (G) Correlation between RNF20 and FASN mRNA expression levels obtained from the TCGA database was calculated using Pearson correlation analysis (inverse correlation). (H) Correlation of RNF20 and ELOVL6 mRNA expression levels obtained from the TCGA database was calculated using Pearson correlation analysis (inverse correlation). (I) qRT-PCR analysis of SREBP2 in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient. (J) shows corrected SREBP2 RNA-seq expression levels in kidney cancer tumors and normal kidney tissues. (K) qRT-PCR analysis of HMGCR in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient. (L) Corrected HMGCR RNA-seq expression levels were shown in renal cancer tumors and normal kidney tissues. * P <0.05; *** P <0.001.
도 11은 신장암 세포에서 RNF20 억제는 지방생합성과 세포증식을 촉진시킨다는 것을 나타낸 것이다. (A) ACHN 신장암 세포주에 siRNF20 또는 siSREBP1을 형질도입하였다. 48시간 후 세포융해물을 SDS-PAGE에서 분리한 후 항체를 이용하여 웨스턴 블랏팅을 수행하였다. nSREBp1, 핵 SREBP1. (B와 C) ACHN 신장암 세포주에 siRNA를 이용하여 RNF20 또는 SREBP1을 녹다운 한 후 지방생합성 관련 유전자들의 mRNA 발현량을 qRT-PCR을 이용하여 분석하였다. mRNA 발현량은 GAPDH mRNA 발현량으로 보정하였고, Mock 대조군에 대한 상대값으로 표현하였다. (D) ACHN 신장암 세포주에 siRNF20 또는 siSREBP1을 형질도입한 후 세포 내 트리글리세리드 양을 측정하였다. (E) ACHN 신장암 세포주에 siRNF20 또는 siSREBP1을 형질도입한 후 세포주기 조절유전자들의 mRNA 발현량을 qRT-PCR을 이용하여 분석하였다. (F) ACHN 신장암 세포주에 siRNF20 또는 siSREBP1을 형질도입한 후 세포증식률을 CCK-8 기법을 통해 측정하였다. 모든 실험은 독립적으로 최소 세 번 수행되었으며 대표 결과를 표현하였다. #P < 0.05 siControl과 비교, ##P < 0.01 siControl과 비교, ###P < 0.001 siControl과 비교, *P < 0.05, **P < 0.01, ***P < 0.001.FIG. 11 shows that RNF20 inhibition in kidney cancer cells promotes lipobiosynthesis and cell proliferation. (A) siRNF20 or siSREBP1 were transduced into ACHN kidney cancer cell lines. After 48 hours, cell lysates were separated on SDS-PAGE and Western blotting was performed using antibodies. nSREBp1, nuclear SREBP1. (B and C) After knocking down RNF20 or SREBP1 by siRNA in ACHN kidney cancer cell lines, mRNA expression levels of adipose biosynthesis-related genes were analyzed using qRT-PCR. mRNA expression level was corrected by GAPDH mRNA expression level and expressed as relative value to Mock control group. (D) After transducing siRNF20 or siSREBP1 to ACHN kidney cancer cell line, the amount of intracellular triglycerides was measured. (E) After transducing siRNF20 or siSREBP1 to ACHN kidney cancer cell lines, mRNA expression levels of cell cycle regulators were analyzed using qRT-PCR. (F) After transduction of siRNF20 or siSREBP1 into ACHN kidney cancer cell lines, cell proliferation was measured by CCK-8 technique. All experiments were performed at least three times independently and represented representative results. # P <0.05 siControl, ## P <0.01 siControl, ### P <0.001 siControl, * P <0.05, ** P <0.01, *** P <0.001.
도 12는 PTTG1은 SREBP1c에 의해 유도되며, 신장암 환자의 종양조직에서 발현량이 증가되어 있다는 것을 나타낸 것이다. (A) 정상 생쥐와 SREBP1c가 결핍된 생쥐의 신장, 간 및 지방 조직을 분리하였다. 각종 조직에서 RNA를 추출한 후 SREBP1c와 PTTG1의 mRNA 발현량을 qRT-PCR을 통해 분석하였다. mRNA 발현량은 TATA-결합 단백질(TBP) 발현량으로 보정하였다. EAT, 내장 지방조직, IAT; 피하 지방조직; BAT; 갈색 지방조직 **P < 0.01, ***P < 0.001. (B) 여러 종에서 PTTG 프로모터에 존재하는 SRE 모티프 및 E-BOX 서열을 도식화한 것이다.Figure 12 shows that PTTG1 is induced by SREBP1c, the expression level is increased in the tumor tissue of kidney cancer patients. (A) Kidney, liver and adipose tissues of normal mice and mice lacking SREBP1c were isolated. After extracting RNA from various tissues, mRNA expression levels of SREBP1c and PTTG1 were analyzed by qRT-PCR. mRNA expression level was corrected by TATA-binding protein (TBP) expression level. EAT, visceral adipose tissue, IAT; Subcutaneous adipose tissue; BAT; Brown adipose tissue ** P <0.01, *** P <0.001. (B) SRE motif and E-BOX sequence present in PTTG promoter in various species.
도 13은 베툴린은 신장암 세포증식을 효과적으로 저하시킨다는 것을 나타낸 것이다. (A와 B) ACHN 신장암 세포주에 siControl과 siRNF20를 형질도입하였다. 베툴린을 24시간 동안 처리한 후 qRT-PCR을 이용하여 지방생합성과 세포주기 조절유전자들의 mRNA 발현량을 측정하였다. mRNA 발현량은 GAPDH 발현량으로 보정하였다. 자료는 독립 표본 세 개의 평균 ± 표준편차를 나타낸다. #P < 0.05 대조군과 비교, ##P < 0.01 대조군과 비교, ###P < 0.001 대조군과 비교, **P < 0.01, ***P < 0.001. (C와 D) (A와 B)에 기술된 신장암 세포주의 성장곡선을 CCK-8 기법을 이용하여 측정하였다. P < 0.05, ***P < 0.001, n.s., 유의미하지 않음; CCK-8, 세포수 계산 키트-8.13 shows that betulin effectively lowers kidney cancer cell proliferation. (A and B) siControl and siRNF20 were transduced into ACHN kidney cancer cell lines. After 24 hours of betulin treatment, qRT-PCR was used to measure mRNA expression of adipose biosynthesis and cell cycle regulators. mRNA expression level was corrected by GAPDH expression level. The data represent the mean ± standard deviation of three independent samples. # P <0.05 compared with control, ## P <0.01 compared with control, ### P <0.001 compared with control, ** P <0.01, *** P <0.001. Growth curves of kidney cancer cell lines described in (C and D) (A and B) were measured using the CCK-8 technique. P <0.05, *** P <0.001, ns, not significant; CCK-8, Cell Counting Kit-8.
도 14는 SREBP1c는 신장암 세포증식을 두 가지 경로(지방생합성과 세포주기 조절)를 통해 조절한다는 것을 나타낸 것이다. (A와 B) ACHN 신장암 세포주에 토파(10μg/ml) 또는 C75(10μg/ml)를 24시간 동안 처리하였다. 지방생합성 및 세포주기 조절유전자들의 mRNA 발현량은 GAPDH mRNA 발현량으로 보정하고 대조군에 대한 상대값으로 표현하였다. (C) (A와 B)에 서술된 세포의 성장속도를 CCK-8 기법을 통해 측정하였다. (D와 E) 렌티바이러스를 통해 SREBP1c를 과발현시킨 ACHN 신장암 세포주에 FASN siRNA를 48시간 동안 처리한 후 qRT-PCR을 이용하여 지방생합성 및 세포주기 조절유전자들의 mRNA 발현량을 측정하였다. 상대적인 mRNA 발현량은 GAPDH mRNA 발현량으로 보정하고 대조군에 대한 상대값으로 표현하였다. #P < 0.05 대조군과 비교, ##P < 0.01 대조군과 비교. (F) (D와 E)에 서술된 세포의 성장속도를 CCK-8 기법을 통해 측정하였다.Figure 14 shows that SREBP1c regulates renal cancer cell proliferation through two pathways (lipid biosynthesis and cell cycle regulation). (A and B) ACHN kidney cancer cell lines were treated with Topa (10 μg / ml) or C75 (10 μg / ml) for 24 hours. MRNA expression of adipose biosynthesis and cell cycle regulators was corrected by GAPDH mRNA expression and expressed as relative to control. (C) The growth rate of the cells described in (A and B) was measured by CCK-8 technique. (D and E) The ACHN kidney cancer cell line overexpressing SREBP1c with a lentiviral was treated with FASN siRNA for 48 hours, and then mRNA expression of adipose biosynthesis and cell cycle regulators was measured using qRT-PCR. Relative mRNA expression was corrected with GAPDH mRNA expression and expressed as relative to control. # P <0.05 compared to control, ## P <0.01 compared to control. (F) The growth rate of the cells described in (D and E) was measured by CCK-8 technique.
도 15는 신장암 이종 이식 종양에서 RNF20는 세포사멸촉진 및 세포사멸억제 유전자를 제어한다는 것을 나타낸 것이다. ACHN 신장암 이종 이식 종양에서 RNF20 과발현이 세포사멸 유전자 발현량 제어에 미치는 영향을 qRT-PCR을 이용하여 분석하였다. 상대적인 mRNA 발현량은 GAPDH mRNA 발현량으로 보정하고 Mock 대조군에 대한 상대값으로 표현하였다. **P < 0.01, ***P < 0.001.15 shows that RNF20 controls apoptosis and apoptosis genes in kidney cancer xenograft tumors. The effect of RNF20 overexpression on apoptotic gene expression control in ACHN kidney cancer xenograft tumors was analyzed using qRT-PCR. Relative mRNA expression was corrected by GAPDH mRNA expression and expressed as relative to the mock control. ** P <0.01, *** P <0.001.
도 16은 간암세포주인 HCC, Huh-7 및 HepG2 세포주에서 RNF20 및 SREBP의 상대적 발현량을 유전자(왼편) 및 단백질(수준)에서 측정한 결과로, 간암세포주에서 대조군(HEK293T)과 비교하여 RNF20의 발현량이 감소되고, SREBP는 증가되어 있음을 나타낸다. FIG. 16 shows the relative expression levels of RNF20 and SREBP in HCC, Huh-7 and HepG2 cell lines, which are liver cancer cell lines, as measured by gene (left) and protein (level), and compared with the control group (HEK293T) in liver cancer cell line. The amount of expression is decreased, and the SREBP is increased.
HEK293T (human embroynic kidney cell; non-cancer cell); Huh-7 (human hepatocellular carcinoma cell; mutant p53-Y220C); HepG2 (human hepatoblastoma cell; wild-type p53). *P < 0.05 vs. HEK293T, **P < 0.01 vs. HEK293T.HEK293T (human embroynic kidney cell; non-cancer cell); Huh-7 (human hepatocellular carcinoma cell; mutant p53-Y220C); HepG2 (human hepatoblastoma cell; wild-type p53). * P <0.05 vs. HEK293T, ** P <0.01 vs. HEK293T.
도 17은 RNF20 발현에 의해 세포 증식이 간암세포주에서 감소되는 것을 나타내는 결과로, 세포주를 RNF20을 발현하는 플라스미드로 전달이입 48시간 후에, CCK-8(cell counting kit-8)를 이용하여 측정하였다. 17 shows that cell proliferation was reduced in hepatocellular carcinoma cell lines by RNF20 expression. The cell lines were measured using CCK-8 (cell counting kit-8) 48 hours after transfer to plasmid expressing RNF20.
도 18은 RNF20 발현에 의해 간암세포주에서 SREBP1c 단백질의 농도가 감소되는 것을 나타내는 웨스턴블랏 결과로, 분석은 세포주를 RNF20을 발현하는 플라스미드로 전달이입 48시간 후에 수행되었다. FIG. 18 is a Western blot showing the decrease of the concentration of SREBP1c protein in liver cancer cell line by RNF20 expression. Analysis was performed 48 hours after delivery of the cell line to the RNF20 expressing plasmid.
도 19는 RNF20 발현에 의해 간암세포주에서 지방생성 유전자의 발현이 감소하는 것을 나타내는 정량 RT-PCR 결과로, 분석은 세포주를 RNF20을 발현하는 플라스미드로 전달이입 48시간 후에 수행되었다. *P < 0.05 vs. Mock, #P < 0.05.Figure 19 is a quantitative RT-PCR results showing that the expression of adipogenetic genes in liver cancer cell lines by RNF20 expression, the analysis was performed 48 hours after delivery of the cell line to the RNF20 expressing plasmid. * P <0.05 vs. Mock, # P <0.05.
도 20은 RNF20의 발현을 siRNF20을 이용하여 억제한 간암세포주에서 세포 증식이 촉진되는 것을 나타내는 결과로, 분석은 세포주를 siRNF20 처리 48시간 후에, CCK-8(cell counting kit-8)를 이용하여 측정하였다. 20 shows that cell proliferation is promoted in liver cancer cell lines in which RNF20 expression is suppressed using siRNF20. Assays are measured using CCK-8 (cell counting kit-8) after 48 hours of siRNF20 treatment. It was.
도 21은 RNF20의 발현을 siRNF20을 이용하여 억제한 간암세포주에서 SREBP1 단백질 농도가 증가하는 것을 나타내는 결과로, 분석은 세포주를 siRNF20 처리 72시간 후에 수행되었다. FIG. 21 shows that SREBP1 protein concentration was increased in liver cancer cell lines which inhibited the expression of RNF20 using siRNF20. Analysis was performed 72 hours after treatment with siRNF20.
도 22는 RNF20의 발현을 siRNF20을 이용하여 억제한 간암세포주에서 지방생성 유전자의 발현이 증가하는 것을 나타내는 정량 RT-PCR 결과로, 분석은 세포주를 siRNF20 처리 72시간 후에 수행되었다. *P < 0.05, **P < 0.01.Figure 22 is a quantitative RT-PCR results showing the increase in the expression of adipogenic genes in hepatocarcinoma cell lines that inhibited the expression of RNF20 using siRNF20, the analysis was performed 72 hours after treatment with the cell line siRNF20. * P <0.05, ** P <0.01.
본원은 신장암 및 간암 환자의 종양 조직에서 RNF20(Ring finger protein 20)의 발현량이 저하되고, RNF20의 과발현이 간암 또는 신장암 세포주 및 이종이식 동물 모델에서 간암 및 신장암 세포의 증식 및 지방대사 활성을 억제하며, 나아가 RNF20이 핵심 조절인자로서, RNF20-SREBP1c-PTTG1 및 RNF20-SREBP1c-지방생합성 경로를 조절하는 것을 규명하였다. The present application shows that the expression level of ring finger protein 20 (RNF20) is decreased in tumor tissues of renal and liver cancer patients, and the overexpression of RNF20 is associated with the proliferation and fat metabolism activity of liver and kidney cancer cells in liver or kidney cancer cell lines and xenograft models. In addition, RNF20 as a key regulator, it was found to regulate the RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipid biosynthesis pathway.
이에 한 양태에서 본원은 RNF20(Ring finger protein 20) 유전자 또는 그 단백질 또는 이와 기능적으로 동등한 변이체, 또는 RNF20 유전자의 메틸화억제를 통해 그 발현을 증가시키는 물질을 포함하는 신장암 또는 간암의 예방 또는 치료용 조성물에 관한 것이다. In one aspect, the present application for the prevention or treatment of kidney cancer or liver cancer, including a ring finger protein 20 (RNF20) gene or a protein or a functional equivalent thereof, or a substance that increases the expression through methylation inhibition of the RNF20 gene To a composition.
본원에 따른 조성물에 포함되는 RNF20(Ring finger protein 20)은, 전사조절, DNA 손상 반응, 줄기세포 분화 및 지방생합성에 다양한 역할을 하는 E3 유비퀴틴 리가아제의 한 종류로서, 크로마틴 리모델링에 기여하는 히스톤 H2B의 모노유비퀴틴화를 촉진시킨다(Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10, 483-488 ; 상기 Shema et al., 2011). Ring finger protein 20 (RNF20) included in the composition according to the present invention is a type of E3 ubiquitin ligase that plays a variety of roles in transcriptional regulation, DNA damage response, stem cell differentiation and lipogenesis, and contributes to histone remodeling. Promote monoubiquitination of H2B (Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10 , 483-488; Shema et al., 2011, supra).
본원에 따른 조성물에 포함되는 RNF20의 유전자 또는 단백질 서열은 본원에 따른 효과를 달성하는 한 다양한 형태 및 유래의 것이 사용될 수 있다. 일 구현예에서는 포유류, 특히 인간 유래의 RNF20 또는 이와 기능적으로 동등한 변이체가 사용되며, 그 유전자 및 단백질 서열은 각각 NCBI Entrez Gene ID: 56254 및 34878777로 공지되어 있다. 본원에 따른 일 구현예에서는 각각 서열번호 1 및 2이 RNF 유전자 및 단백질 서열이 사용된다. The gene or protein sequence of RNF20 included in the composition according to the present invention can be used in various forms and derived so long as the effect according to the present application is achieved. In one embodiment RNF20 or a functionally equivalent variant from mammals, in particular humans, is used, the genes and protein sequences of which are known as NCBI Entrez Gene ID: 56254 and 34878777, respectively. In one embodiment according to the invention SEQ ID NO: 1 and 2, respectively, the RNF gene and protein sequence is used.
본원에 따른 유전자는 게놈 DNA, cDNA 및 화학합성 DNA를 포함한다. 게놈 DNA 및 cDNA는 당업계의 공지된 방법에 따라 제조할 수 있다. 게놈 DNA는 예를 들어, 해당 유전자를 가지는 세포로부터 게놈 DNA를 추출하고 게놈 라이브러리(벡터는 예를 들면 플라스미드, 파지, 코스미드, BAC, PAC 등이 이용될 수 있다)를 제작하고 상기 라이브러리를 본 발명의 단백질을 암호화하 는 DNA(예를 들어, 서열번호 2)를 기초로 제작한 프로브를 이용하여 콜로니 또는 플라그 혼성화를 수행하거나, 본 발명의 단백질을 암호화하는 DNA(예를 들어, 서열번호 2)에 특이적인 프라이머를 작성하고, 이를 이용한 PCR(Polymerase Chain Reaction)을 행함으로써 제조할 수도 있다. cDNA는 예를 들어, RNF20 유전자를 가지는 세포에서 추출한 mRNA를 기초로 cDNA를 합성하고, 상기 합성된 cDNA를 λZAP 등의 벡터로 삽입하여 cDNA 라이브러리를 제작하고, 상기 cDNA 라이브러리를 전개하여, 상기와 동일하게 콜로니 또는 플라그 혼성화를 수행하거나 또는 PCR을 수행하여 제조할 수 있다.Genes according to the present invention include genomic DNA, cDNA and chemical synthetic DNA. Genomic DNA and cDNA can be prepared according to methods known in the art. Genomic DNA, for example, extracts genomic DNA from cells having the gene of interest and constructs a genomic library (vectors may be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and viewed the library. Colony or plaque hybridization is performed using a probe made on the basis of DNA encoding the protein of the invention (eg, SEQ ID NO: 2), or DNA encoding the protein of the present invention (eg, SEQ ID NO: 2). It can also be prepared by preparing a primer specific for the) and performing PCR (Polymerase Chain Reaction) using the same. For example, cDNA synthesizes a cDNA based on mRNA extracted from a cell having an RNF20 gene, inserts the synthesized cDNA into a vector such as λZAP, prepares a cDNA library, and expands the cDNA library. Can be prepared by colony or plaque hybridization or by PCR.
본원에서 용어 “기능적으로 동등한 변이체”란 특정 종 유래의 야생형 단백질 또는 이의 유전자 서열과 비교하여 변이가 있지만 본원에서 규명한 효능을 갖는 것을 말한다. 따라서, 예를 들면 본원발명의 RNF20 단백질 또는 이를 코딩하는 유전자는 사람 유래의 서열로서, 하나 이상의 아미노산이 치환, 결실, 부가 및/또는 삽입된 아미노산 서열로 이루어지는 단백질을 암호화하는 돌연변이체(mutants), 유도체(derivatives), 대립유전자(alleles), 변형체(variants) 및 동질체 (homologues)를 포함한다. 나아가, 예를 들어, 염기서열의 변이가 단백질 중의 아미노산의 변이를 수반하지 않는 경우(축중변이)도 있고, 이러한 축중변이체(degeneracy mutants)도 본 발명의 유전자에 포함된다.As used herein, the term "functionally equivalent variant" refers to a variant compared to a wild type protein or gene sequence thereof derived from a particular species, but having the efficacy identified herein. Thus, for example, the RNF20 protein of the present invention or a gene encoding the same may be a mutants encoding a protein derived from a human sequence consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, Derivatives, alleles, variants and homologues. Furthermore, for example, variations in nucleotide sequences may not be accompanied by variations in amino acids in proteins (degeneracy variants), and such degeneracy mutants are also included in the gene of the present invention.
RNF20 단백질 및 이와 기능적으로 동등한 단백질을 암호화하는 유전자는 당업계의 공지된 방법, 예를 들면 PCR 방법(Saiki et al., Science, 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988)을 이용하여 제조될 수 있다. 예를 들면, 당업자라면 공지된 RNF20 유전자의 염기서열로부터 이에 특이적으로 혼성화할 수 있는 프라이머를 고안하여 RNF20 유전자와 높은 상동성을 가지는 다양한 포유류 유래의 유전자를 분리할 수 있을 것이다. The RNF20 protein and the gene encoding the functionally equivalent protein are known in the art, for example PCR methods (Saiki et al., Science, 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988). For example, those skilled in the art will be able to isolate primers from a variety of mammals having high homology with the RNF20 gene by designing primers capable of specific hybridization thereof from known RNF20 gene sequences.
상기와 같이 분리된 유전자는 아미노산 수준에 있어서, 인간 유래 RNF20 단백질의 아미노산 서열과 높은 상동성을 가진다. 높은 상동성이란 아미노산 서열 전체에서, 적어도 50% 이상, 더 바람직하게는 70% 이상, 더 바람직하게는 90%이상(예를 들어, 95% 이상)의 서열의 동일성을 가리킨다. 아미노산 서열이나 염기서열의 상동성은 BLAST(Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993)에 기초하여 개발된 BLASTN 이나 BLASTX라 불리는 프로그램(Altschul et al, J. Mol. Biol., 215, 403-410, 1990)을 사용하여 분석될수 있으며 구체적인 방법은 다음의 웹사이트에 공지되어 있다(http://www,ncbi.nlm.nih.gov.).The gene isolated as described above has high homology with the amino acid sequence of the human-derived RNF20 protein at the amino acid level. High homology refers to the identity of at least 50%, more preferably at least 70%, more preferably at least 90% (eg, at least 95%) sequences throughout an amino acid sequence. The homology of amino acid sequences or nucleotide sequences is based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993), a program called BLASTN or BLASTX (Altschul et al, J. Mol. Biol. , 215, 403-410, 1990) and specific methods are known on the following website (http: //www,ncbi.nlm.nih.gov.).
본원에 따른 조성물에 포함되는 RNF20 단백질 또는 유전자는 전장 또는 잘린 형태의 것이 모두 포함된다. 또한 동일한 숙주, 예를 들면 인간에서 유래된 것이라도 특정 개인, 지역, 환경 등에 따라 서열변이가 있을 수 있으며, 이러한 것은 물론 일부 서열이 변형(결실, 치환, 부가)되었으나, 기능적으로 동등한 변이체가 모두 본 발명에 사용될 수 있다. 한 구현예에서는 인간 유래의 것으로 이에 관하여는 앞서 언급한 바와 같다.The RNF20 protein or gene included in the composition according to the present invention includes all of full length or truncated forms. In addition, there may be sequence variations according to a specific individual, region, environment, etc., even if they are derived from the same host, for example, humans, and of course, some sequences have been modified (deleted, substituted, added), but all functionally equivalent variants are present. It can be used in the present invention. In one embodiment it is of human origin and is as previously mentioned.
본원에 따른 조성물이 RNF20 유전자 또는 이와 동등한 변이체를 포함하는 경우, 상기 단백질을 코딩하는 유전자는 재조합 DNA 기술 등 공지된 방법에 따라, 표적세포 예를 들면 포유류 유래의 종양세포, 특히 신장암 세포에서 RNF20 유전자를 발현할 수 있는 한 이로 제한되는 것은 아니나, 예를 들면, 선형 DNA, 플라스미드 벡터 또는 그 외 DNA 전달용 벡터로 도입되어 사용될 수 있다. 이러한 벡터는 바람직하게는 비바이러스성이며, 진핵세포에서 RNF20 유전자를 발현할 수 있는 한, 특별히 제한되는 것은 아니며, 본원 실시예에 기재된 것을 참고할 수 있다. When the composition according to the present invention comprises an RNF20 gene or equivalent variant thereof, the gene encoding the protein is RNF20 in a target cell, for example, a tumor cell of mammalian origin, in particular a kidney cancer cell, according to known methods such as recombinant DNA technology. As long as the gene can be expressed, but not limited thereto, for example, it may be introduced and used as a linear DNA, a plasmid vector or other DNA delivery vector. Such vectors are preferably non-viral and are not particularly limited as long as they can express the RNF20 gene in eukaryotic cells, and reference may be made to those described in the Examples herein.
또한 본 발명의 RNF20 유전자는 유전자치료 시스템 등에 사용되는 발현벡터 예를 들면 바이러스벡터에 도입되어 공지된 방법에 따라 전달체로서 바이러스입자에 포함되어 사용될 수 있다. 상기의 바이러스 벡터는 본 발명의 단백질을 목적하는 세포 또는 조직내로 도입할 수 있는 것이면 제한되지 않으며 바람직하게는 아데노바이러스 벡터이며, 상기 바이러스성 벡터는 진핵세포에서 이에 포함된 유전자를 발현할 수 있는 한, 특별히 제한되는 것은 아니다. In addition, the RNF20 gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, for example, a viral vector, and then included in a viral particle as a carrier according to a known method. The viral vector is not limited as long as the protein of the present invention can be introduced into a desired cell or tissue, and is preferably an adenovirus vector, and the viral vector is capable of expressing a gene contained therein in a eukaryotic cell. It is not particularly limited.
본원에 따른 조성물이 단백질 또는 이와 기능적으로 동등한 변이체를 포함하는 경우, 예를 들면 상기 벡터를 공지된 방법에 따라 세포배양 시스템에서 적절한 세포에 트렌스펙션하여 발현시킨 후 단백질을 정제하여 사용할 수 있다. 이러한 본 발명의 단백질은 예를 들면 정제된 단백질, 수용성 단백질, 또는 표적 세포로의 전달 또는 투여를 위해 담체에 결합된 형태의 단백질 또는 아미노산 잔기와 융합된 형태의 것을 포함하는 것이다.When the composition according to the present invention comprises a protein or a functionally equivalent variant thereof, for example, the vector may be purified and used after transfection into an appropriate cell in a cell culture system according to a known method. Such proteins of the invention include, for example, purified proteins, water soluble proteins, or those in fusion with protein or amino acid residues in a form bound to a carrier for delivery or administration to a target cell.
본원에 따른 조성물에 포함되는 RNF20 발현을 증가시키는 물질은 상기 유전자, 특히 내인성 유전자 즉 세포 자체가 원래부터 가지고 있는 유전자의 프로모터 부위의 메틸화를 억제하는 물질로, 특히 CpG 섬에서의 메틸화를 억제하는 물질로, 메틸화의 억제에 의해 RNF20의 전사가 촉진되고, 결국 RNF20의 세포에서의 발현이 증가하게 된다. 따라서 이러한 효과를 나타내는 다양한 물질이 본원에 따른 조성물에 포함될 수 있다. 일 구현예에서는 DNMT (DNA methyltransferase) 억제제가 사용된다. DNMT (DNA methyltransferase) 억제제는 후생적 유전자 조절 약물로서 뉴클레오사이드 유사체로서 예를 들면 azacytidine, decitabine 및 zebularine을 포함하나, 이로 제한되는 것은 아니며, 상술한 효과를 포함하는 다양한 물질이 포함될 수 있다. 또한 비-뉴클레오사이드 유사체가 사용될 수 있으며, 이는 hydralazine, RG109, procainnamide, procaine, SCI-1027 등을 들 수 있다. 비-뉴클레오사이드 유사체는 DNMT가 CpG 섬에 결합하는 것을 방해하여 결국 메틸화를 억제하며, 이 과정에서 DNMT와 특이적으로 상호작용을 한다. 본원에 따른 일 구현예에서는 RG108이 사용된다. Substances that increase the expression of RNF20 included in the composition according to the present application is a substance that inhibits methylation of the promoter region of the gene, particularly an endogenous gene, that is, the cell itself, in particular, a substance that inhibits methylation in CpG islands. Inhibition of methylation promotes the transcription of RNF20, resulting in increased expression of RNF20 in cells. Therefore, various materials which exhibit this effect can be included in the compositions according to the present application. In one embodiment a DNA methyltransferase (DNMT) inhibitor is used. DNA methyltransferase (DNMT) inhibitors include, but are not limited to, nucleoside analogs as epigenetic gene-modulating drugs such as, for example, azacytidine, decitabine, and zebularine, and may include various substances including the effects described above. Non-nucleoside analogs may also be used, including hydralazine, RG109, procainnamide, procaine, SCI-1027, and the like. Non-nucleoside analogs interfere with the binding of DNMT to the CpG island, which in turn inhibits methylation, and in the process specifically interacts with DNMT. In one embodiment according to the present application RG108 is used.
본원에 따른 조성물이 사용되는 신장암은 신장의 실질(신장에서 소변을 만드는 세포들이 모여 있는 부분으로 수질과 피질로 구성됨)에서 발생하는 신장세포암을 포함하며, 투명세포형 신세포암(clear cell RCC, ccRCC), 유두형 신세포암(papillary RCC), 혐색소형 신세포암(chromophobe RCC), 수질형 신세포암(medullary RCC), 분류불능 신세포암(unclassified RCC), 신이행상피암(kidney transitional cell carcinoma, TCC), 신장 호산성과립세포종(ranal oncocytoma)을 포함한다. 일구현예에서는 ccRCC이다. Kidney cancer in which the composition according to the present invention is used includes renal cell carcinoma that occurs in the parenchyma of the kidney (consisting of the medulla and the cortex as a collection of urine-forming cells in the kidney), and clear cell type renal cell carcinoma (clear cell cancer) RCC, ccRCC), Papillary RCC, Chromophobe RCC, Medullary RCC, Unclassified RCC, Kidney transitional cancer cell carcinoma (TCC), renal eosinophilic granuloma (ranal oncocytoma). In one embodiment, it is ccRCC.
본원에 따른 조성물이 사용되는 간암은 간세포암(Hepatocellular carcinoma, HCC)이다. 이는 알콜 남용, 바이러스성 간염 및 대상성 간질환과 같은 위험인자를 갖는 환자에서 발생하는 간조직 자체에서 발생하는 원발성 악성 종양을 일컫는 것이다. Liver cancer in which the composition according to the present invention is used is hepatocellular carcinoma (HCC). This refers to primary malignant tumors that occur in the liver tissue itself that occurs in patients with risk factors such as alcohol abuse, viral hepatitis, and target liver disease.
또한 본원에 따른 조성물은 신장암의 VHL (von Hippel-Lindau) 변이 또는 상태와 무관하게 효과를 나타내며, 따라서 보다 다양한 원인의 신장암의 치료 및 진단에 사용될 수 있다. In addition, the composition according to the present invention exhibits an effect irrespective of von Hippel-Lindau (VHL) variation or condition of kidney cancer, and thus can be used for the treatment and diagnosis of kidney cancer of more various causes.
현재까지 알려진 신장암의 주요 발병원인은 VHL 종양억제유전자의 결손 및 돌연변이에 따른 HIF 전사인자의 활성화로 알려져 있다(Nat Rev Cancer. 2008 Nov;8(11):865-73.). 그럼에도 불구하고 신장-특이적 VHL 결핍 생쥐의 경우 신장암 발병과 유사한 형질이 나타나지 않았으며(Cancer Res. 2006 Mar 1;66(5):2576-83.), HIF1이 과활성화된 신장암 세포의 이종 이식 동물 모델에서 종양 증식이 예상과 달리 억제된다는 결과가 보고되었다(J Mol Med (Berl). 2014 Aug;92(8):825-36.). 이상의 연구결과는 신장암 발병 기전에 VHL-HIF 경로 이외에 별도의 핵심 분자기전의 존재를 나타내는 것이며, 바로 본원에서 RNF20-SREBP1c 경로가 VHL 유전자와 결손/돌연변이와는 독립적으로 신장암 발생에 관여하는 것을 규명하였다. The major cause of kidney cancer known to date is the activation of HIF transcription factors due to deletion and mutation of the VHL tumor suppressor gene (Nat Rev Cancer. 2008 Nov; 8 (11): 865-73.). Nevertheless, renal-specific VHL deficient mice did not show similar traits as renal cancer development (Cancer Res. 2006 Mar 1; 66 (5): 2576-83.), And HIF1 overactivated kidney cancer cells. It was reported that tumor proliferation was unexpectedly inhibited in xenograft animal models (J Mol Med (Berl). 2014 Aug; 92 (8): 825-36.). These findings indicate the presence of a key molecular mechanism in addition to the VHL-HIF pathway in the pathogenesis of renal cancer. Herein, the RNF20-SREBP1c pathway is involved in renal cancer development independently of the VHL gene and deletion / mutation. It was clarified.
따라서 본원의 조성물 및 방법은 VHL 변이와 독립적으로 신장암의 치료 및 진단에 사용될 수 있다. 일 구현예에서는 본원의 조성물 및 방법이 사용되는 신장암은 VHL 변이를 포함하거나 또는 포함하지 않을 수 있다. 다른 구현예에서는 본원의 조성물 및 방법은 VHL 변이가 원인이 아닌 신장암에 사용될 수 있다. Thus, the compositions and methods herein can be used for the treatment and diagnosis of kidney cancer independently of VHL mutations. In one embodiment, the kidney cancer in which the compositions and methods herein are used may or may not include VHL variations. In other embodiments, the compositions and methods herein can be used in kidney cancer that is not caused by VHL mutations.
본원에서 사용된 용어 “치료”란 질환, 또는 질환으로 인한 증상 또는 상태의 억제, 제거, 경감, 완화, 개선, 및/또는 예방을 포함하는 개념이다.As used herein, the term “treatment” is a concept that includes inhibiting, eliminating, alleviating, alleviating, ameliorating, and / or preventing a disease or symptom or condition resulting from the disease.
본원의 조성물은 RNF20 이외에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 또는 유효성분의 용해성 및/또는 흡수성을 유지/증가시키는 화합물을 추가로 함유할 수 있다. 또한 본원의 약학 조성물은 신장암의 치료 또는 예방을 위하여 단독으로, 또는 수술, 약물치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present application may further contain a compound which maintains / increases the solubility and / or the absorbency of at least one active ingredient or an active ingredient exhibiting the same or similar function in addition to RNF20. In addition, the pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of kidney cancer.
본원의 조성물은 상기 언급한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 씨딩 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술분야의 적정한 방법으로 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제형화할 수 있다.The composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, seeding, and one or more of these components, as necessary. Other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.
본원의 조성물의 투여방법은 특별히 이에 제한되는 것은 아니며, 공지된 투여방법을 적용할 수 있으며, 목적하는 방법에 따라 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 비경구 투여의 경우 피부에 붙이는 패치형, 코/호흡기를 통해 투여할 수 있으며, 신속한 치료효과를 얻기 위해서는 정맥내 주사에 의한 투여가 바람직하다. 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여 시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 매우 다양할 수 있다. RNF20 유전자 또는 폴리펩타이드를 포함한 단백질 제제의 경우 비경구 투여가 선호될 수 있으나 다른 경로 및 수단을 배제하는 것은 아니다. 전형적인 약물의 경우 투약단위체는, 예를 들어 약 0.01mg 내지 100mg를 포함하나 상기 범위의 이하 및 이상의 범위를 배제하는 것은 아니다. 일일 투여량은 약 1μg 내지 10g 일 수 있으며, 하루 일회 내지 수회에 나누어 투여할 수 있다.The method of administration of the composition of the present application is not particularly limited thereto, and known administration methods may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) or oral, depending on the desired method. In the case of parenteral administration, it can be administered through a patch-type, nasal / respirator attached to the skin, and administration by intravenous injection is preferable to obtain a rapid therapeutic effect. The dosage may vary widely depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Parenteral administration may be preferred for protein preparations comprising the RNF20 gene or polypeptide, but does not exclude other routes and means. For typical drugs, dosage units include, for example, about 0.01 mg to 100 mg but do not exclude the below and above ranges. The daily dose may be about 1 μg to 10 g, and may be administered once to several times a day.
다른 양태에서 본원은 RNF20의 바이오마커로서의 용도에 관한 것으로 특히 이의 검출용 물질을 포함하는 간암 또는 신장암의 진단 또는 예후 측정용 조성물 또는 키트와 관련된 것이다. In another aspect the present application relates to the use of RNF20 as a biomarker, and more particularly to a composition or kit for diagnosing or prognosticting liver or kidney cancer comprising a substance for detection thereof.
본원에서 용어 진단은 특정 질병 또는 질환에 대하여 검사 대상자의 질환에 대한 감수성(susceptibility)을 판정하는 것, 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 대상자의 예후(prognosis)(예컨대, 트랜지션성 암 상태의 동정, 암의 단계 또는 진행상태 판별 또는 치료에 대한 암의 반응성 결정)를 판정하는 것 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 포함한다.As used herein, the term diagnosis refers to determining the susceptibility of a subject's disease to a particular disease or condition, determining whether he or she currently has a particular disease or condition, prognosis of the subject having a particular disease or condition ( prognosis (eg, identifying a transitional cancer state, determining stage or progression of a cancer, or determining the responsiveness of a cancer to treatment) or to determine therametrics (eg, to provide information about treatment efficacy). Monitoring the state of an object).
본원에서 용어 진단용 바이오마커 또는 진단 마커(diagnosis marker)란 암 발생한 조직 또는 세포를 정상 세포 또는 적절한 치료를 받은 조직 또는 세포와 구분하여 진단할 수 있는 물질로, 정상 검체에 비하여 질환이 발생한 조직이나 부위에서 증가 또는 감소 양상을 보이는 단백질 또는 핵산을 포함한다. 본원에서 신장암 진단 마커는 신장암 조직에서 발현이 감소하는 RNF20 단백질 또는 이를 코딩하는 핵산 또는 유전자이다.As used herein, the term diagnostic biomarker or diagnostic marker is a substance that can diagnose cancer-causing tissues or cells from normal cells or appropriately treated tissues or cells and diagnose the diseased tissues or regions as compared to normal samples. Protein or nucleic acid showing an increase or decrease in Renal cancer diagnostic marker herein is an RNF20 protein or a nucleic acid or gene encoding the same that reduces expression in kidney cancer tissue.
본원에서 생물학적 시료란 바이오마커 검출이 가능한 하나 이상의 성분을 포함하는 물질 또는 물질의 혼합물을 일컫는 것으로 생물체, 특히 인간 유래의 세포, 조직 또는 체액, 예를 들면 전혈, 혈장, 및 혈청을 포함하나 이로 제한하는 것은 아니다. 또한 생물체에서 직접적으로 유래된 것은 물론 인비트로에서 배양된 세포 또는 조직을 포함한다. 본원에 따른 간암 또는 신장암 마커의 검출을 위해 다양한 시료가 사용될 수 있으나, 이로 제한하는 것은 아니다. 한 구현예에서는 전혈, 혈청 및/또는 혈장이 사용될 수 있다. 다른 구현예에서는 간암 또는 신장암이 발생한 또는 발생이 의심되는 또는 발생가능성이 있는 생물체에서 수득한 신조직/세포 또는 인비트로 세포 배양물이 사용될 수 있으나, 이로 제한하는 것은 아니다. 또한 상기 혈액, 세포 또는 조직의 분획 또는 유도물을 포함하는 것이다. 세포 또는 조직을 이용하는 경우, 세포 자체 또는 세포 또는 조직의 융해물이 사용될 수 있다. Biological sample as used herein refers to a substance or mixture of substances that includes one or more components capable of detecting a biomarker and includes, but is not limited to, cells, tissues or body fluids, such as whole blood, plasma, and serum from an organism, in particular humans. It is not. It also includes cells or tissues derived directly from an organism as well as cultured in vitro. Various samples may be used for the detection of liver or kidney cancer markers according to the present disclosure, but are not limited thereto. In one embodiment, whole blood, serum and / or plasma may be used. In other embodiments, renal tissue / cells or in vitro cell cultures obtained from, or suspected of developing or likely to develop, liver cancer or kidney cancer may be used. It also includes fractions or derivatives of the blood, cells or tissues. When using a cell or tissue, the cell itself or a fusion of the cell or tissue may be used.
본원에서 RNF20 단백질 및 그 핵산 서열은 공지된 것이며, 또한 앞서 언급한 바와 같으나, 이로 한정하는 것은 아니며, 이의 기능적 동등체를 포함하는 것이다. The RNF20 protein and nucleic acid sequences thereof are known herein and are also as previously mentioned, but not limited to, including functional equivalents thereof.
본원에서 검출이란, 정량 및/또는 정성 분석을 포함하는 것으로, 존재, 부존재의 검출 및 발현량 검출을 포함하는 것으로 이러한 방법은 당업계에 공지되어 있으며, 당업자라면 본원의 실시를 위해 적절한 방법을 선택할 수 있을 것이다.Detection herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level detection. Such methods are well known in the art and those skilled in the art will select appropriate methods for carrying out the present application. Could be.
본원에 따른 마커는 정량적 또는 정성적 분석을 통해 핵산, 특히 mRNA 및/또는 단백질의 존재 여부의 검출 및/또는 이의 발현량 자체, 발현량의 변화, 발현량 차이의 수준에서 검출될 수 있다.The markers according to the invention can be detected at the level of the detection of the presence of nucleic acids, in particular of mRNA and / or protein, and / or their expression levels themselves, changes in expression levels, differences in expression amounts, through quantitative or qualitative analysis.
이러한 본원에 따른 간암 또는 신장암 진단용 마커는 이의 기능적 특징 및/또는 항원적 특징에 기반을 둔 것이다. 단백질의 활성, 기능 또는 단백질을 코딩하는 핵산, 특히 mRNA 및/또는 단백질 수준(level)에서 특이적으로 상호작용하는 제제를 사용하여 검출될 수 있다. Such markers for diagnosing liver or kidney cancer according to the present application are based on their functional and / or antigenic characteristics. The activity, function or activity of a protein can be detected using agents that specifically interact at the nucleic acid encoding the protein, particularly at the mRNA and / or protein level.
이런 측면에서 본원은 또한 각 바이오 마커의 핵산서열, 상기 핵산서열에 상보적인 핵산서열, 상기 핵산서열의 단편, 또는 상기 핵산서열에 의해 코딩되는 단백질을 특이적으로 인식하는 항체 또는 앱타머를 포함하는 신장암 진단용 마커에 관한 것이다.In this aspect, the present application also includes an antibody or aptamer specifically recognizing a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence, or a protein encoded by the nucleic acid sequence. It relates to a marker for diagnosing kidney cancer.
본원에서 용어 검출용 물질은 본원의 마커를 단백질 또는 핵산 예를 들면 mRNA 수준에서 정량적으로 또는 존재여부의 판단을 위해 검출할 수 있는 물질이다. As used herein, the term detecting substance is a substance capable of detecting the marker of the present invention quantitatively or for the presence of a protein or nucleic acid such as mRNA level.
단백질의 양, 존재여부 발현패턴의 분석을 위한 방법은 공지된 것으로 예를 들면 웨스턴 블롯, ELISA(enzyme linked immunosorbent assay), 방사선 면역분석(Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테를로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역 전기영동, 조직 면역 염색, 면역 침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), FACS, 단백질 칩(protein chip) 등을 들 수 있다. 단백질 또는 핵산 수준검출을 위한 시약은 공지된 것으로서, 예를 들면 전자는 항원-항체반응, 상기 마커에 특이적으로 결합하는 기질, 핵산 또는 펩타이드 앱타머, 상기 마커와 특이적으로 상호작용하는 수용체 또는 리간드 또는 보조인자와의 반응을 통해 검출될 수 있거나, 또는 질량분광분석기를 이용할 수 있다. 상기 본원의 마커와 특이적으로 상호작용 또는 결합하는 시약 또는 물질은 칩 방식 또는 나노입자(nanoparticle)과 함께 사용될 수 있다.Methods for the analysis of the amount and presence of the protein expression patterns are known, for example Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, oukterroni Ouchterlony) immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like. Reagents for detecting protein or nucleic acid levels are known, for example, the former may be an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor that specifically interacts with the marker or It can be detected through reaction with a ligand or cofactor, or a mass spectrometer can be used. Reagents or materials that specifically interact with or bind to the markers of the present disclosure may be used with chip or nanoparticles.
본원에 따른 진단 조성물은 RNF20 마커를 단백질 또는 핵산 예를 들면 mRNA 수준에서의 검출에 필요한 시약을 포함할 수 있다. 예를 들면 상기 단백질 수준에서 검출할 수 있는 시약은 모노클로날 항체, 폴리클로날 항체, 기질, 앱타머, 수용체, 리간드 또는 보조인자 등을 포함할 수 있다. 이러한 시약은 필요한 경우 나노입자 또는 칩에 통합하여 사용할 수 있다. 단백질은 또한 질량분광분석기를 이용하여 검출될 수 있다.The diagnostic composition according to the present disclosure may comprise reagents necessary for the detection of an RNF20 marker at a protein or nucleic acid such as at the mRNA level. For example, reagents detectable at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands, cofactors, and the like. Such reagents can be incorporated into nanoparticles or chips as needed. Proteins can also be detected using mass spectrometry.
RNA 수준에서의 검출, 발현량 또는 패턴의 검출을 위해 전사 중합효소연쇄반응(RT-PCR)/중합효소연쇄반응, 경쟁적 RT-PCR, 실시간 RT-PCR RNase 보호 분석법, 칩 또는 노던블랏 등을 이용한 방식이 사용될 수 있으며, 이러한 분석법은 공지된 것이며, 또한 시중의 키트를 사용하여 수행될 수 있으며, 당업자라면 본원의 실시를 위해 적절한 것을 선택할 수 있을 것이다. 예를 들면 상기 mRNA의 존재 여부와 그 양 또는 패턴을 RT-PCR로 측정하기 위한 방법에서 검출시약으로, 예를 들면 본원 마커의 mRNA에 특이적인 프라이머를 포함한다. 프라이머는 주형과 상보적으로 결합할 수 있고 역전사효소 또는 DNA 중합효소가 주형의 복제를 개시할 수 있도록 하는 자유 3말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열을 의미한다.Detection of RNA levels, expression levels or patterns using transcription polymerase chain reaction (RT-PCR) / polymerase chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assays, chips or Northern blots Methods can be used, and such assays are known and can also be performed using commercial kits, and one skilled in the art will be able to select the appropriate one for the practice herein. For example, as a detection reagent in a method for measuring the presence and amount or pattern of the mRNA by RT-PCR, for example, a primer specific for mRNA of the marker of the present application is included. By primer is meant a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template.
본원에 사용되는 상기 검출 시약은 신호검출을 위해 형광물질과 같은 발색물질과 컨쥬게이션 된 것일 수 있다.The detection reagent used herein may be conjugated with a coloring material such as a fluorescent material for signal detection.
본원에서 검출이란, 정량 및/또는 정성 분석을 포함하는 것으로, 존재, 부존재의 검출 및 발현량 검출을 포함하는 것으로 이러한 방법은 당업계에 공지되어 있으며, 당업자라면 본원의 실시를 위해 적절한 것 방법을 선택할 수 있을 것이다. 원의 일 구현예에 따르면 검출시약은 항체를 포함하며, 본원의 RNF20 단백질 검출은 이에 특이적으로 결합하는 모노클로날 항체를 이용하여 실시된다.Detection herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level detection. Such methods are well known in the art, and those skilled in the art will recognize methods suitable for the practice of the present application. You will be able to choose. According to one embodiment of the original detection reagent comprises an antibody, the RNF20 protein detection of the present application is carried out using a monoclonal antibody that specifically binds thereto.
본원에 이용될 수 있는 항체는 폴리클로날 또는 모노클로날 항체이며, 바람직하게는 모노클로날 항체이다. 항체는 당업계에서 통상적으로 실시되는 방법들, 예를 들어, 융합 방법(Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)), 재조합 DNA 방법(미국 특허 제4,816,56호) 또는 파아지 항체 라이브러리 방법(Clackson et al, Nature, 352:624-628(1991) 및 Marks et al, J. Mol. Biol., 222:58, 1-597(1991))에 의해 제조될 수 있다. 항체 제조에 대한 일반적인 과정은 Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; 및 Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991에 상세하게 기재되어 있으며, 상기 문헌들은 본 명세서에 참조로 삽입된다. 예를 들어, 단일클론 항체를 생산하는 하이브리도마 세포의 제조는 불멸 세포주를 항체-생산 림프구와 융합시켜 이루어지며, 이 과정에 필요한 기술은 당업자에게 잘 알려져 있으며 용이하게 실시할 수 있다. 폴리클로날 항체는 단백질 항원을 적합한 동물에게 주사하고, 이 동물로부터 항혈청을 수집한 다음, 공지의 친화성(affinity) 기술을 이용하여 항혈청으로부터 항체를 분리하여 얻을 수 있다.Antibodies that can be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortal cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.
이러한 면역분석은 종래에 개발된 다양한 정량적 또는 정성적 면역분석 프로토콜에 따라 실시될 수 있다. 상기 면역분석 포맷은 방사능면역분석, 방사능면역침전, 면역침전, 면역조직화학염색, ELISA(enzyme-linked immunosorbant assay), 캡처-ELISA, 억제 또는 경쟁 분석, 샌드위치 분석, 유세포 분석(flow cytometry), 면역형광염색 및 면역친화성 정제를 포함하지만, 이에 한정되는 것은 아니다. 상기 면역분석 또는 면역염색의 방법은 Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay(ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984 등에 기재되어 있으며, 상기 문헌은 본 명세서에 참조로서 삽입된다. 상술한 면역분석 과정에 의한 최종적인 시그널의 세기를 분석하여 즉, 정상 시료와의 시그널 대조를 수행함으로써, 질환 발생 여부를 진단할 수 있다.Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunity. Including but not limited to fluorescent staining and immunoaffinity purification. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984, etc., which is incorporated herein by reference. By analyzing the final signal intensity by the above-described immunoassay process, that is, by performing a signal contrast with a normal sample, it is possible to diagnose whether the disease occurs.
본원의 다른 구현예에 따르면 검출시약은 RNA 분석용 시약으로, 본원에 따른 검출은 mRNA 수준에서 실시된다. mRNA 검출은 통상 노던블랏이나 역전사 PCR (중합효소연쇄반응)을 통해 수행된다. 후자의 경우 검체의 RNA를 특히 mRNA를 분리한 후, 이로부터 cDNA를 합성한 후, 특정 프라이머, 또는 프라이머 및 프로브의 조합을 사용하여, 검체 중의 특정 유전자를 검출하는 것으로, 특정 유전자의 존재/부존재 또는 발현량을 결정할 수 있는 방법이다. 이러한 방법은 예를 들면 (Han, H.; Bearss, D. J.; Browne, L. W.; Calaluce, R.; Nagle, R. B.; Von Hoff, D. D., Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002, 62, (10), 2890-6)에 기재되어 있다.According to another embodiment of the invention the detection reagent is a reagent for RNA analysis, the detection according to the present application is carried out at the mRNA level. mRNA detection is usually carried out by Northern blot or reverse transcriptase PCR (polymerase chain reaction). In the latter case, the RNA of a sample is specifically isolated from mRNA, and then cDNA is synthesized therefrom, and then a specific gene or a combination of primers and probes is used to detect a specific gene in the sample. Or it is a method which can determine the expression amount. Such methods are described, for example, in (Han, H .; Bearss, DJ; Browne, LW; Calaluce, R .; Nagle, RB; Von Hoff, DD, Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002 , 62, (10), 2890-6).
본원에서는 RNF20가 저발현되는 간암 및 신장암 세포주 또는 이를 이식한 마우스에서 RNF20의 과발현은 SREBP1c 발현을 감소시키고, 이는 다시 PTTG1의 발현을 감소시켜, 신장암 세포주의 사멸을 증가시키고, 증식은 억제하는 것을 발견하였다. 나아가 상기 SREBP1c 발현을 감소는 간암 및 신장암 세포에서 지질대사에 관여하는 유전자의 발현의 감소로 이어지고, 암세포의 증식이 억제되는 것을 발견하였다. Herein, overexpression of RNF20 in liver and kidney cancer cell lines or mice transplanted with low expression of RNF20 reduces SREBP1c expression, which in turn decreases the expression of PTTG1, thereby increasing the death of kidney cancer cell lines and inhibiting proliferation. I found that. Furthermore, it was found that decreasing the expression of SREBP1c leads to a decrease in the expression of genes involved in lipid metabolism in liver and kidney cancer cells and inhibits the proliferation of cancer cells.
따라서 다른 양태에서 본원은 RNF20-SREBP1c(Sterol Regulatory Element Binding Transcription Factor 1c)-PTTG1(Pituitary Tumor Transforming 1) 또는 RNF20-SREBP1c-지방생합성 촉진 단백질 신호전달 경로를 표적으로 하는 RNF20의 발현 이상과 관련된 암 치료제 스크리닝 방법에 관한 것이다. Thus, in another embodiment, the present application provides a therapeutic agent for cancer associated with abnormal expression of RNF20 targeting RNF20-SREBP1c (Sterol Regulatory Element Binding Transcription Factor 1c) -PTTG1 (Pituitary Tumor Transforming 1) or RNF20-SREBP1c-lipidogenesis promoting protein signaling pathway. It relates to a screening method.
본원에서는 RNF20의 저발현 또는 발현 억제 또는 결여가 신장암 또는 간암의 발생과 연관되어 있으며, 또 그 기전을 규명한 것으로 RNF20의 발현 이상 특히 저발현 또는 결여가 암의 발생으로 이어진 경우라면, 다양한 암에 대한 치료제가 본원의 스크리닝 방법에 의해 발굴될 수 있다. 이러한 암의 예로는 특히 신장암, 또는 간암을 포함하는 이로 제한하는 것은 아니다. 일 구현예에서 신장암, 특히 VHL (von Hippel-Lindau) 유전자 변이가 원인이 아닌 신장암으로, 상술한 바와 같이 이러한 신장암은 VHL 변이를 포함할 수도 있고, 하지 않을 수도 있으며, VHL 변이를 포함하는 경우에도 이러한 변이가 신장암의 원인이 아니다. In the present application, the low expression or suppression or lack of expression of RNF20 is associated with the development of renal cancer or liver cancer, and elucidated the mechanism of the various cancers, if abnormal expression of RNF20, in particular, low expression or lack of, led to the development of cancer. Therapeutic agents for can be discovered by the screening methods herein. Examples of such cancers are not particularly limited to those including kidney cancer or liver cancer. In one embodiment kidney cancer, particularly kidney cancer that is not caused by von Hippel-Lindau (VHL) gene mutations, as described above, may or may not include VHL mutations and includes VHL mutations. Even if these mutations are not the cause of kidney cancer.
본원에 따른 방법은 일 구현예에서 RNF20의 발현이 감소 또는 결여된 세포를 제공하는 제 1 단계; 상기 세포를 RNF20의 발현을 증가시킬 것으로 기대되는 시험물질로 처리하는 제 2 단계; 상기 시험물질로 처리된 세포에서 상기 SREBP1c 또는 PTTG1 중 하나 이상의 발현을 측정하는 제 3 단계; 및 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 SREBP1c 또는 PTTG1 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 SREBP1c 또는 PTTG1의 발현이 상기 대조군의 SREBP1c 또는 PTTG1 발현과 비교하여 감소한 경우, 이를 RNF20의 발현 이상과 관련된 암의 치료제 후보물질로 선별하는 제 4 단계를 포함한다. The method according to the present invention, in one embodiment, comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase expression of RNF20; A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And comparing the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance and the control cells not in contact with the test substance, so that the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance is less than that of the control. And a fourth step of selecting it as a therapeutic agent candidate for cancer associated with abnormal expression of RNF20.
본원에 따른 방법에서 RNF20의 발현의 감소 또는 결여는 RNF20 유전자 자체의 변이 또는 후생적 조절에 의해 단백질로의 발현이 감소 또는 억제되었거나, 또는 유전자의 이상을 수반하지 않는 경우라도 발현된 단백질이 후생적 조절 등에 의해 목적하는 기능을 하지 못하는 경우를 포함하는 것으로, 당업자라면, 정상 세포 및 본원의 실시예에 기재된 것을 참조하여 발현이 감소 또는 억제된 정도를 판단할 수 있을 것이다. 예를 들면 RNF20의 유전자 발현(또는 전사)이 억제되어 단백질 발현(또는 번역) 감소로 이어진 경우, 또는 단백질로 발현되었으나, 유전자 변이 등으로 인해 단백질이 목적하는 기능을 하지 못하는 경우, 유전자가 메틸화되어 유전자의 발현이 억제된 경우 등을 포함한다. 결여된 경우의 예로는 RNF20의 유전자는 존재하나, 메틸화 등의 후생적 조절에 의해 발현되지 않는 경우를 포함한다. In the method according to the present invention, the reduction or lack of expression of RNF20 is reduced or inhibited by the mutation or epigenetic regulation of the RNF20 gene itself, or the expressed protein is epigenetic even if it does not involve abnormality of the gene. Including the case where the desired function is not performed by regulation or the like, those skilled in the art will be able to determine the extent to which expression is reduced or inhibited with reference to normal cells and those described in the Examples herein. For example, if gene expression (or transcription) of RNF20 is inhibited, leading to a decrease in protein expression (or translation), or if it is expressed as a protein but the protein does not function as desired due to gene mutation, the gene is methylated. And when the expression of the gene is suppressed. Examples of cases lacking include the case where the gene of RNF20 is present but not expressed by epigenetic regulation such as methylation.
본원에 따른 방법에서는 RNF20의 발현이 감소 또는 결여된 세포로는 신장 또는 간암 조직 유래의 세포 또는 세포주가 사용된다. RNF20의 발현은 유전자 및/또는 단백질 수준에서의 감소 또는 결여를 의미하는 것으로, 발현의 감소 또는 결여는 정상 신장 세포주를 대조군으로 사용하여 이와 비교하여 감소된 것으로 당업자면 본원의 개시사항 및 당업계의 수준을 근거로 감소된 수준을 판단할 수 있다. 일 구현예에서 본원에 따른 방법에 사용되는 RNF20의 발현이 감소된 세포는 신장 세포주로 ACHN, A498, 또는 Caki-2, 간암 세포주로는 Huh-7 또는 HepG2를 포함하나 이로 제한하는 것은 아니다. In the method according to the present invention, cells or cell lines derived from renal or hepatic cancer tissue are used as the cells with reduced or lacking the expression of RNF20. Expression of RNF20 refers to a decrease or lack in gene and / or protein levels, wherein a decrease or lack of expression is reduced in comparison to using a normal kidney cell line as a control. Based on the level, the reduced level can be judged. In one embodiment the cells with reduced expression of RNF20 used in the methods according to the present disclosure include, but are not limited to, ACHN, A498, or Caki-2 as a kidney cell line, Huh-7 or HepG2 as a liver cancer cell line.
일 구현예에서 상기 방법은 RNF20의 발현이 감소 또는 결여된 세포를 제공하는 제 1 단계; 상기 세포의 RNF20의 발현을 증가시키거나 또는 SREBP1c 발현을 억제시킬 것으로 기대되는 시험물질로 처리하는 제 2 단계; 상기 시험물질로 처리된 세포에서 PTTG1의 발현을 측정하는 제 3 단계; 및 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 PTTG1 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 PTTG1의 발현이 상기 대조군의 PTTG1 발현과 비교하여 감소한 경우 이를 신장암 치료제 후보물질로 선별하는 제 4 단계를 포함한다. In one embodiment the method comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating with a test substance expected to increase the expression of RNF20 or inhibit SREBP1c expression of said cells; A third step of measuring expression of PTTG1 in cells treated with the test substance; And comparing the PTTG1 expression in the cells contacted with the test substance and the control cells not in contact with the test substance, and when the expression of the PTTG1 in the cell contacted with the test substance decreased compared with the PTTG1 expression of the control. And a fourth step of selecting a candidate for treating a kidney cancer.
다른 구현예에서는 상기 제 3 단계에서, 상기 SREBP1c의 발현을 측정하는 대신에, 또는 이에 부가하여, 상기 SREBP1c에 의해 발현이 촉진되는 지질생합성에 관여하는 유전자 또는 그 단백질의 발현을 측정하는 단계를 포함하며, 이 경우 상기 제 4 단계에서 상기 후보물질은 상기 시험물질과 접촉된 세포에서 상기 시험물질과 접촉되지 않은 대조군 세포와 비교하여 상기 지질생합성에 관여하는 유전자 또는 단백질 발현이 감소한 것이다. In another embodiment, in the third step, instead of or in addition to measuring the expression of SREBP1c, measuring the expression of a gene or a protein thereof involved in lipid biosynthesis in which expression is promoted by the SREBP1c. In this case, in the fourth step, the candidate material has a decreased expression of a gene or protein involved in the lipid biosynthesis in comparison with control cells not in contact with the test material in cells in contact with the test material.
또 다른 구현예에서는 상기 제 3 단계 대신에, 상기 SREBP1c에 의해 발현이 증가, 향상 또는 촉진되는 지질생합성에 관여하는 단백질 및/또는 그 유전자의 발현을 측정하는 단계를 포함하며, 그리고 상기 제 4 단계 대신에, 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 지질생합성에 관여하는 단백질 및/또는 유전자의 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 지질생합성에 관여하는 단백질의 발현이 상기 대조군의 지질생합성에 관여하는 단백질의 발현과 비교하여 감소한 경우 이를 신장암 치료제 후보물질로 선별하는 단계를 포함한다. In another embodiment, instead of the third step, measuring the expression of a protein and / or a gene involved in lipid biosynthesis in which expression is increased, enhanced, or promoted by the SREBP1c, and the fourth step Instead, the expression of the proteins and / or genes involved in the lipid biosynthesis in the cells in contact with the test substance and in control cells not in contact with the test substance was compared to determine the lipid biosynthesis in the cells in contact with the test substance. If the expression of the involved protein is reduced compared to the expression of the protein involved in the lipid biosynthesis of the control group comprising the step of selecting it as a candidate for the treatment of kidney cancer.
본원에 따른 방법에서 RNF20-SREBP1c-지질생합성 촉진 경로를 발견한 것으로, 본원에서 지질생합성에 관여하는 유전자 또는 그 단백질이란 SREBP1c에 의해 그 전사가 증가되고, 또한 단백질로의 발현이 증가되는 것으로, 발현증가에 의해 세포에서 지질의 합성이 증가 또는 촉진된다. 따라서 이러한 특징을 갖는 한 특별히 제한하는 것은 아니나, 신장암에서는 지질대사에 관여하는 물질은 FAS(Fatty acid synthase, 또는 유전자는 FASN), SCD1(Stearoyl-CoA desaturase-1), 또는 ELOVL6(Elongation of very long chain fatty acids protein 6)의 유전자 또는 단백질의 발현을 측정하여 선별할 수 있다. 상기 각 단백질 및 그 유전자 서열은 예를 들면 FASN NCBI 유전자 ID (2194), 단백질 DB (41872631); ACC1 유전자 ID (31), 단백질 DB (38679960); SCD1 유전자 ID (6319), 단백질 DB (53759151); 및 ELOVL6 유전자 ID (79071), 단백질 DB (195539343)로 공지된 것으로 당업자라면, 상기 서열 및 본원에 개시된 것을 근거로 상기 단백질 또는 유전자의 발현을 용이하게 측정할 수 있을 것이다. 또한 상기 유전자 이외에 지질 생합성에 관여하는, SREBP1c의 표적 유전자로서 공지된 다른 유전자의 측정을 배제하는 것은 아니다. 측정 결과 상기 시험물질과 접촉된 세포에서 상기 지질대사에 관여하는 단백질의 발현이 상기 대조군의 지질대사에 관여하는 단백질의 발현과 비교하여 감소한 경우 이를 신장암 치료제 후보물질로 선별할 수 있다. In the method according to the present invention, RNF20-SREBP1c-lipid biosynthesis promoting pathway was found, wherein the gene or the protein involved in lipid biosynthesis is that its transcription is increased by SREBP1c and its expression to the protein is increased. The increase increases or promotes the synthesis of lipids in the cell. Therefore, as long as these characteristics are not particularly limited, substances that are involved in lipid metabolism in kidney cancer are FAS (Fatty acid synthase, or gene is FASN), SCD1 (Stearoyl-CoA desaturase-1), or ELOVL6 (Elongation of very Long chain fatty acids protein 6) can be selected by measuring the expression of the gene or protein. Said each protein and its gene sequence are for example FASN NCBI gene ID (2194), protein DB (41872631); ACC1 Gene ID (31), Protein DB (38679960); SCD1 gene ID (6319), protein DB (53759151); And ELOVL6 Gene ID (79071), Protein DB (195539343), will be readily apparent to those skilled in the art, based on the sequences and disclosed herein, the expression of the protein or gene. It is also not intended to exclude the measurement of other genes known as target genes of SREBP1c that are involved in lipid biosynthesis in addition to these genes. As a result of the measurement, when the expression of the protein involved in the lipid metabolism in the cell contacted with the test substance decreases compared with the expression of the protein involved in the lipid metabolism of the control group, it may be selected as a candidate for treating cancer of the kidney.
본원에 따른 방법에서 분자 표적은 RNF20 및/또는 SREBP1c로서, 전자의 발현을 증가시키는 물질 또는 후자의 발현을 감소시키는 물질은 신장암 치료제 스크리닝의 시험물질로 사용될 수 있다. In the method according to the present invention, the molecular targets are RNF20 and / or SREBP1c, and the substance which increases the expression of the former or the substance which decreases the expression of the latter can be used as a test substance for screening a therapeutic agent for kidney cancer.
본원의 방법에 사용되는 시험물질은 상술한 표적 유전자 또는 단백질의 발현 및/또는 활성을 조절할 것으로 기대되는 물질로, 예를 들면 약물의 스크리닝 목적을 위해서는 화합물은 저분자량의 치료효과를 갖는 것이 사용될 수 있다. 예를 들면 중량이 400 Da, 600 Da 또는 800 Da과 같은 약 1000 Da 내외의 화합물이 사용될 수 있다. 목적에 따라 이러한 화합물은 화합물 라이브러리의 일부를 구성할 수 있으며, 라이브러리를 구성하는 화합물의 숫자도 수십개부터 수백만개까지 다양하다. 이러한 화합물 라이브러리는 펩타이드, 펩토이드 및 기타 환형 또는 선형의 올리고머성 화합물, 및 주형을 기본으로 하는 저분자 화합물, 예컨대 벤조디아제핀, 하이단토인, 바이아릴, 카보사이클 및 폴리사이클 화합물(예컨대 나프탈렌, 페노티아진, 아크리딘, 스테로이드 등), 카보하이드레이트 및 아미노산 유도체, 디하이드로피리딘, 벤즈하이드릴 및 헤테로사이클(예컨대 트리아진, 인돌, 티아졸리딘 등)을 포함하는 것일 수 있으나, 이는 단지 예시적인 것으로 이로 한정되는 것은 아니다. The test substance used in the method of the present invention is a substance which is expected to modulate the expression and / or activity of the target gene or protein described above. For example, for the purpose of screening a drug, the compound may have a low molecular weight therapeutic effect. have. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely illustrative. It is not limited to this.
상기와 같은 시험물질의 처리에 의한 RNF20의 발현을 증가시키거나 또는 SREBP1c 발현을 억제는 상기 세포에서 PTTG1의 발현을 측정하여 결정될 수 있다. PTTG1는 그 서열이 공개된 것으로 인간 cDNA 서열은 GenBank ID: NM_004219, 단백질 서열은 NP_001269311를 참조할 수 있다. PTTG1의 발현은 단백질 또는 유전자 수준에서 검출될 수 있으며, 이에 대하여는 앞서 기술한 바를 참고할 수 있다. Increasing the expression of RNF20 or suppressing SREBP1c expression by treatment of such test substance can be determined by measuring the expression of PTTG1 in the cells. PTTG1 has disclosed its sequence, and may refer to GenBank ID: NM_004219 for human cDNA sequence and NP_001269311 for protein sequence. Expression of PTTG1 can be detected at the protein or gene level, for which reference can be made above.
후보물질은 상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 PTTG1 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 PTTG1의 발현이 상기 대조군의 PTTG1 발현과 비교하여 감소한 경우 이를 신장암 치료제 후보물질로 선별할 수 있다. Candidates compared the PTTG1 expression in cells in contact with the test substance and control cells not in contact with the test substance, whereby the expression of the PTTG1 in the cells in contact with the test substance decreased compared to the PTTG1 expression of the control. If this can be selected as a candidate candidate for the treatment of kidney cancer.
또한 상기와 같은 시험물질의 처리에 의한 RNF20의 발현을 증가시키거나 또는 SREBP1c 발현을 억제는 상기 세포에서 지질대사에 관여하는 단백질의 발현을 측정하여 결정될 수 있다. In addition, increasing the expression of RNF20 or suppressing SREBP1c expression by the treatment of the test substance can be determined by measuring the expression of proteins involved in lipid metabolism in the cells.
본 방법에서 사용되는 세포의 종류 및 시험물질의 양 및 종류 등은 사용하는 구체적인 실험방법 및 시험물질의 종류에 따라 달라지며, 당업자라면 적절한 양을 선택할 수 있을 것이다. 실험결과 시험물질과 접촉되지 않은 대조군과 비교하여 시험물질의 존재 하에서 상기 단백질 발현 또는 활성의 감소를 가져오는 물질을 후보 물질로 선별한다. 대조군과 비교 약 99% 이상 감소, 약 95% 이상 감소, 약 90% 이상 감소, 약 85% 이상 감소, 약 80% 이상 감소, 약 75% 이상 감소, 약 70% 이상 감소, 약 65% 이상 감소, 약 60% 이상 감소, 약 55% 이상 감소, 약 50% 이상 감소, 약 45% 이상 감소, 약 40% 이상 감소, 약 30% 이상 감소, 약 20% 이상 감소, 약 10% 이상 감소를 의미하나, 이를 벗어나는 범위를 제외하는 것은 아니다.The type of cell used in the present method and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, as a candidate, a substance which results in a decrease in the expression or activity of the protein in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance. Compared with the control group, about 99% reduction, about 95% reduction, about 90% reduction, about 85% reduction, about 80% reduction, about 75% reduction, about 70% reduction, about 65% reduction , About 60% reduction, about 55% reduction, about 50% reduction, about 45% reduction, about 40% reduction, about 30% reduction, about 20% reduction, about 10% reduction However, it does not exclude the range beyond this.
또 다른 양태에서 본원은 간암 또는 신장암의 진단 또는 예후에 필요한 정보를 제공하기 위하여, 검사 대상체 유래의 생물학적 시료로부터 RNF20 바이오마커의 핵산 및/또는 단백질의 수준을 검출하는 단계; 상기 핵산 및/또는 단백질 수준 검출결과를 대조군 시료의 해당 마커의 상응하는 결과와 비교하는 단계; 및 상기 대조군 시료와 비교하여, 상기 대상체 유래 시료의 핵산 또는 단백질 수준에 변화가 있는 경우, 상기 대상체의 신장암 진단 또는 생존 예후와 연관시키는 단계를 포함하는, RNF20 바이오마커를 검출하는 방법에 관한 것이다. In another aspect, the present disclosure provides a method for detecting a cancer or kidney cancer, comprising: detecting a level of nucleic acid and / or protein of a RNF20 biomarker from a biological sample from a test subject to provide information necessary for the diagnosis or prognosis of liver or kidney cancer; Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And when there is a change in the nucleic acid or protein level of the subject-derived sample compared to the control sample, correlating with a renal cancer diagnosis or survival prognosis of the subject. .
본원에 따른 방법에 사용되는 마커의 발현양의 검출은 단백질 및/또는 mRNA 발현 수준에서 결정될 수 있으며, 이에 관해서는 언급한 바와 같다. 또한 대상체 생물학적 시료는 앞서 언급한 바를 참조할 수 있다. Detection of the amount of expression of a marker used in the method according to the invention can be determined at the protein and / or mRNA expression level, as mentioned above. Subject biological samples may also refer to the foregoing.
본원에 따른 방법에서 상기 연관시키는 단계에서, 상기 대조군은 정상 대조군이며, 상기 정상 대조군에서 결정된 마커의 수준과 비교하여 상기 마커의 수준이 상기 대상체에서 감소한 경우 상기 대상제를 신장암 또는 생존 예후가 좋지 않은 것으로 예측할 수 있다. 생존 예후란 신장암으로 진단되어 치료 후에 5년 생존율을 일컫는 것으로, 병리학적, 임상적 및 분자생물학적 소견을 종합하여 결정할 수 있다. 생존률은 특히 암의 병기와도 관련성이 있으며, 신장암의 병기에 따른 일반적으로 제 1기는 88-100%, 제 2기는 63-88%, 제 3기는 34-59% 그리고 제 4기는 0-20%이며, 이와 비교하여 낮은 것을 의미한다. In the step of associating in the method according to the present invention, the control group is a normal control group, and when the level of the marker decreases in the subject compared to the level of the marker determined in the normal control group, the subject agent has a poor renal cancer or a poor survival prognosis. Can be predicted. Survival prognosis refers to 5-year survival after diagnosis of kidney cancer, which can be determined by combining pathological, clinical and molecular biological findings. Survival rates are especially associated with cancer stages, which are generally 88-100% in stage 1, 63-88% in stage 2, 34-59% in stage 3 and 0-20 in stage 4 %, Which means lower than that.
판단을 위해 대조군 예를 들면 정상 대조군 또는 완치된 대조군의 값과 비교하여 예를 들면 RNF20이 유의하게 감소된 경우, 대상체에서 상기 질환이 발생한 것으로 진단하는 정보를 제공할 수 있다. 본원 일 구현예에 따르면 상기 연관시키는 단계는 정상 대조군과 대상체의 시료를 비교한 후, 상기 각 마커에 대하여 발병여부를 진단할 수 있는 임계값을 설정한 후, 대상체의 검출 결과를 상기 임계값과 비교할 수 있다. 임계값 설정은 예를 들면 본원 실시예에 기재된 방법을 참조할 수 있다.For the purpose of determining, for example, when the RNF20 is significantly reduced compared to the value of a control group, for example, a normal control or a cured control group, information for diagnosing that the disease has occurred in a subject may be provided. According to an embodiment of the present disclosure, the step of associating a sample of the subject with a normal control group, sets a threshold value for diagnosing the onset of each marker, and then detects the detection result of the subject with the threshold value. Can be compared. Threshold settings may refer to, for example, the methods described in the Examples herein.
본원의 방법은 간암 또는 신장암의 진단 또는 예후에 관한 정보를 제공하기 위해, 마커 분석 결과에 추가하여, 환자의 비단백질 임상정보 즉, 마커 이외의 임상정보를 추가로 사용할 수 있다. 이러한 비단백질 임상정보란, 예를 들면 환자의 나이, 성별, 체중, 식습관, 체질량, 기저질환, 초음파, 전산화단층촬영(CT) 중 하나 이상을 포함하나, 이로 제한하는 것은 아니다.The methods herein can further use non-protein clinical information, i.e., non-marker clinical information, of the patient in addition to marker analysis results to provide information regarding the diagnosis or prognosis of liver or kidney cancer. Such nonprotein clinical information includes, but is not limited to, for example, one or more of age, sex, weight, diet, body mass, underlying disease, ultrasound, computed tomography (CT) of the patient.
또 다른 양태에서 본원은 인비트로 또는 동물에서 RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현 증가를 가져오는 물질을 이용한 세포의 RNF20-SREBP1c-PTTG1 신호전달 경로 조절 방법 또는 키트에 관한 것이다. In another embodiment, the present disclosure provides RNF20-SREBP1c-PTTG1 signaling in cells using a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that results in increased expression of the gene in vitro or in an animal. A route control method or kit.
또 다른 양태에서 본원은 또한 인비트로 또는 동물에서 RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현 증가를 가져오는 물질을 이용한 세포의 RNF20-SREBP1c-지질생합성 경로 조절 방법 또는 키트에 관한 것이다. In another aspect, the present disclosure also provides RNF20-SREBP1c-lipid biosynthesis of cells using a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that results in increased expression of the gene in vitro or in an animal. A route control method or kit.
상기 키트 및 방법에 사용되는 물질 및 이를 이용한 조절 기전에 대한 설명은 앞서 언급한 바를 참조할 수 있다. 본원에 따른 방법 및 키트는 신장 또는 간암의 치료, 기존의 약물 테스트, 신약 개발, 연구툴로서 유용하게 사용될 수 있으나, 이로 제한 하는 것은 아니다. For a description of the materials used in the kits and methods and the regulatory mechanism using the same, reference may be made to the foregoing. The methods and kits according to the present application may be usefully used as treatments for kidney or liver cancer, existing drug tests, drug development, research tools, but are not limited thereto.
이하, 본 발명의 이해를 돕기 위해서 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.
실시예 Example
실험방법Experiment method
세포배양 및 화합물Cell Culture and Compound
ACHN, A498, HEK293, Caki-2 및 정상 신장 피질 표피(HRCE; human primary renal cortical epithelial) 세포주, HEK293T, Huh-7 및 HepG2는 American Type Culture Collection(ATCC)에서 획득하였고, 공급자의 메뉴얼대로 배양되었다. 요약하면 ACHN과 A498 세포주는 10% 소태아혈청(FBS; fetal bovine serum), 페니실린(100U/ml), 스트렙토마이신(100μg/ml)을 첨가한 Eagle’s minimum essential medium(MEM) 배지에서 배양하였다. HEK293과 Caki-2 세포주는 10% FBS와 페니실린, 스트렙토마이신이 첨가된 Dulbecco’s modified Eagle medium(DMEM) 배지에서 배양하였다. HRCE 세포주는 0.5% FBS, 10 nM 트라이아이오도타이로닌, 10ng/ml 표피생장인자, 100ng/ml 하이드로코티손, 5μg/ml 인슐린, 1μM 에피네프린, 5μg/ml 트랜스페린, 2.4 mM L-알라닌-L-글루타민, 페니실린과 스트렙토마이신을 첨가한 신장 표피세포 배양배지에서 배양하였다. 모든 세포는 5% 이산화탄소, 37℃ 조건에서 배양하였다. 베툴린(Betulin) 및 보디피(BODIPY) 493/503은 시그마-알드리치를 통해 구입하였다. C75 및 TOFA는 Abcam에서 구입하였다. 프로피디움 아이오딘화합물은 BD 바이오사이언스에서 구입하였다.ACHN, A498, HEK293, Caki-2 and human primary renal cortical epithelial (HRCE) cell lines, HEK293T, Huh-7 and HepG2 were obtained from the American Type Culture Collection (ATCC) and cultured according to the supplier's manual . In summary, ACHN and A498 cell lines were cultured in Eagle's minimum essential medium (MEM) medium containing 10% fetal bovine serum (FBS), penicillin (100 U / ml) and streptomycin (100 μg / ml). HEK293 and Caki-2 cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) medium containing 10% FBS, penicillin and streptomycin. HRCE cell line 0.5% FBS, 10 nM triiodothyronine, 10 ng / ml epidermal growth factor, 100 ng / ml hydrocortisone, 5 μg / ml insulin, 1 μM epinephrine, 5 μg / ml transferrin, 2.4 mM L-alanine-L-glutamine Cultured in renal epidermal cell culture medium containing penicillin and streptomycin. All cells were cultured in 5% carbon dioxide, 37 ℃ conditions. Betulin and BODIPY 493/503 were purchased via Sigma-Aldrich. C75 and TOFA were purchased from Abcam. Propidium iodine compounds were purchased from BD Biosciences.
인간 신장암 및 간암 조직 샘플Human Kidney and Liver Cancer Tissue Samples
간암 및 신장암 환자의 종양 조직과 정상 신장 조직 표본은 서울대학교병원에서 제공하였다. 서울대학교병원 내 심의기관에서 본 연구를 승인하였다(승인번호: H-1501-011-636). 본 연구의 후향적 특성상 환자의 실험 동의서는 필요하지 않았다. Tumor tissue and normal kidney tissue specimens of liver and kidney cancer patients were provided by Seoul National University Hospital. The study was approved by a review institution at Seoul National University Hospital (approved number: H-1501-011-636). Due to the retrospective nature of this study, the patient's consent was not required.
TCGA RNA-Seq 및 생존 분석TCGA RNA-Seq and Survival Analysis
VHL 돌연변이 상태 및 환자의 임상 자료는 The Cancer Genome Atlas(TCGA)에서 2015년 9월에 획득하였다. 533개의 신장암 종양 조직 및 72개의 정상 신장 샘플에 대한 RNA 서열분석 결과를 box plot 분석은 1-99번째 백분위수(막대), 25-75번째 백분위수(상자) 및 중간값(상자 내 선)을 나타내었다. 생존 분석을 위하여 TCGA RNA-Seq 자료에서 얻은 유전자 발현량에 따라 환자의 순위를 매겼다. 유전자 발현 량이 평균 이상인 환자는 ‘높은’ 집단, 나머지를 ‘낮은’ 집단으로 정의하였다. 전반 생존 곡선은 카플란-마이어 생존 분석을 통해 추정하였으며, 두 집단의 생존 차이는 로그-랭크 분석을 통해 비교하였다.VHL mutation status and patient clinical data were obtained in September 2015 from The Cancer Genome Atlas (TCGA). RNA sequencing results for 533 kidney cancer tumor tissues and 72 normal kidney samples were analyzed using the 1-99th percentile (bar), 25-75th percentile (box), and median (line within the box). Indicated. Patients were ranked according to gene expression from TCGA RNA-Seq data for survival analysis. Patients with higher than average gene expression were defined as "high" and the rest as "low". Overall survival curves were estimated by Kaplan-Meier survival analysis and survival differences between the two groups were compared by log-rank analysis.
조직 마이크로어레이 및 면역조직화학법Tissue Microarrays and Immunohistochemistry
신장암 환자의 종양 조직 및 정상 신장 조직 절편에 대한 면역조직화학법은 제조사의 설명서에 따라 시행되었다. 간략하게 설명하면 조직 마이크로어레이에는 50개의 신장암 종양 조직 절편과 9개의 정상 신장 조직이 포함되었다. 면역조직화학 분석에서 RNF20와 SREBP1을 검출하기 위해 스트렙타비딘-바이오틴 복합체 기법을 사용하였으며, 각각의 항체는 Abcam과 BD 바이오사이언스에서 구입하였다.Immunohistochemistry for sections of tumor tissue and normal kidney tissue from kidney cancer patients was performed according to the manufacturer's instructions. Briefly, the tissue microarray included 50 kidney cancer tumor tissue sections and 9 normal kidney tissues. In the immunohistochemical analysis, the streptavidin-biotin complex technique was used to detect RNF20 and SREBP1, and each antibody was purchased from Abcam and BD Biosciences.
재조합 아데노바이러스 제작Recombinant Adenovirus Production
아데노바이러스 플라스미드는 이전 논문에 보고한대로 제작되었다 (1). 간단히 설명하면, 랫 SREBP1c 아미노산 1-403번과 생쥐 RNF20 전체를 암호화하는 cDNA를 AdTrack-CMV 셔틀 벡터에 클로닝하였고 Ad-Easy 아데노바이러스 벡터 시스템을 통해 제작하였다. 모든 실험에서 GFP를 암호화하는 아데노바이러스를 대조군으로 사용하였다. 아데노바이러스는 HEK293A 세포주에서 증식시켰으며, 기존에 보고된 CsCl 밀도구배원심법을 이용하여 정제하였다 (2).Adenovirus plasmids were constructed as reported in previous papers (1). Briefly, cDNAs encoding rat SREBP1c amino acids 1-403 and the entire mouse RNF20 were cloned into an AdTrack-CMV shuttle vector and constructed via an Ad-Easy adenovirus vector system. In all experiments, adenovirus encoding GFP was used as a control. Adenovirus was propagated in HEK293A cell line and purified using the previously reported CsCl density gradient centrifugal method (2).
렌티바이러스 제작 및 바이러스 형질도입Lentivirus Production and Virus Transduction
Flag 표지가 달린 RNF20와 SREBP1c cDNA를 렌티바이러스 벡터 pLVX-EF1α-AcGFP1-N1에 클로닝하였다. 리포펙타민 2000을 이용하여 렌티바이러스 벡터와 pAX2 및 pMD2.G 벡터를 HEK293T 세포주에 형질도입하였다. 형질도입 48시간 후에 바이러스를 수집하여 0.45-μm 필터를 통해 불순물을 걸러냈다. 이후 ACHN 세포주에 바이러스와 8μg/ml의 폴리브렌을 함유한 배양액에 18시간 동안 배양하였다. 감염된 세포를 48시간 동안 회복시킨 후 퓨로마이신에 저항하는 콜로니들만 선별하여 실험에 이용하였다.Flag labeled RNF20 and SREBP1c cDNA were cloned into the lentiviral vector pLVX-EF1α-AcGFP1-N1. The lentiviral vector and pAX2 and pMD2.G vectors were transduced into HEK293T cell line using lipofectamine 2000. 48 hours after transduction, the virus was collected and filtered through a 0.45-μm filter. Thereafter, the cells were incubated for 18 hours in a culture medium containing 8 μg / ml polybrene and virus in an ACHN cell line. Infected cells were recovered for 48 hours and only colonies resistant to puromycin were selected for use in the experiment.
웨스턴 블랏팅 분석Western blotting analysis
세포 및 조직을 150 mM 염화나트륨, 50 mM 트리스-황산(pH 7.4), 1% NP-40, 0.25% 소듐디옥시콜레이트, 1 mM EDTA, 1 mM PMSF, and 단백질분해효소 억제제 혼합물을 함유한 radioimmunoprecipitation assay(RIPA) 버퍼에 용해시켰다. 각 시료에서 동량의 단백질을 SDS-PAGE 젤에서 분리하였으며, 이후 폴리비닐리덴 다이플루오라이드(PVDF) 막으로 옮겼다. 이후 PVDF 막을 0.1% Tween-20를 함유한 TBS 용액에 5% 무지방 우유 또는 3% 소혈청알부민 첨가하여 비특이반응을 억제하고 RNF20, SREBP1, FASN, SCD1, PTTG1, Cyclin B1, Cyclin E, Myc-tag, Flag-tag 또는 β-actin과 결합하는 항체를 PVDF 막에 결합시켰다. 홀스래디시 과산화효소가 결합된 2차 항체를 PVDF 막에 결합시킨 후, LuminoImager(LAS-3000) 기계를 이용한 화학발광 검출을 통해 단백질을 시각화 하였다.Cells and tissues were radioimmunoprecipitation assay containing 150 mM sodium chloride, 50 mM tris-sulfate, pH 7.4, 1% NP-40, 0.25% sodium dioxycholate, 1 mM EDTA, 1 mM PMSF, and a protease inhibitor mixture. Dissolved in (RIPA) buffer. Equal amounts of protein in each sample were separated on an SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was then added to TBS solution containing 0.1% Tween-20 to suppress nonspecific reactions by adding 5% nonfat milk or 3% bovine serum albumin. Antibodies that bind -tag, Flag-tag or β-actin were bound to the PVDF membrane. After binding the horseradish peroxidase-conjugated secondary antibody to the PVDF membrane, the protein was visualized by chemiluminescence detection using a LuminoImager (LAS-3000) machine.
RNA 추출 및 qRT-PCRRNA extraction and qRT-PCR
전체 RNA를 TRIzol 용해 시약으로 추출하였다. RevertAid 역전사효소를 이용하여 동량의 RNA로부터 cDNA를 합성하였다. 상대적인 mRNA 발현량은 정량적 리얼타임 PCR 분석법(qRT-PCR)을 통해 측정하였으며, GAPDH 또는 Cyclophilin mRNA 양으로 보정한 뒤 계산하였다.Total RNA was extracted with TRIzol lysis reagent. CDNA was synthesized from the same amount of RNA using RevertAid reverse transcriptase. Relative mRNA expression was measured by quantitative real-time PCR analysis (qRT-PCR) and calculated after correction with GAPDH or Cyclophilin mRNA.
[표 1] qRT-PCR의 프라이머 서열TABLE 1 Primer sequences of qRT-PCR
Figure PCTKR2017011172-appb-I000001
Figure PCTKR2017011172-appb-I000001
siRNA 형질도입siRNA transduction
RNA20, SREBP1, PTTG1 및 FASN의 siRNA 이중가닥은 바이오니어(대한민국)에서 합성하였다. 리포펙타민 RNAiMAX 시스템을 사용하여 제공사 설명서에 따라 ACHN 신장암 세포주에 siRNA 형질도입을 실시하였다.SiRNA double strands of RNA20, SREBP1, PTTG1 and FASN were synthesized in Bioneer (South Korea). SiRNA transduction was performed to ACHN kidney cancer cell lines using the lipofectamine RNAiMAX system according to the provider instructions.
[표 2] siRNA Oligos의 서열Table 2 Sequence of siRNA Oligos
Figure PCTKR2017011172-appb-I000002
Figure PCTKR2017011172-appb-I000002
CCK-8을 이용한 세포증식 분석Cell proliferation assay using CCK-8
세포증식은 기존에 보고된 세포수 계산 키트(CCK-8, Cell Counting Kit-8) 시스템을 사용하여 측정하였다 (3). 간단히 설명하면, 살아있는 세포의 대사체를 감지하는 민감비색분석을 수행하여 세포 성장곡선을 얻었다.Cell proliferation was measured using a previously reported Cell Counting Kit-8 (CCK-8) system (3). In brief, cell coloration curves were obtained by performing colorimetric analysis to detect metabolites of living cells.
군집형성능분석Cluster Formation Analysis
RNF20 또는 SREBP1c를 과발현하는 렌티바이러스를 지닌 ACHN 세포주를 6-well 판에서 배양하였다. 세포를 5% 이산화탄소, 37℃ 조건에서 7일간 배양한 후 포름알데히드로 고정하고 크리스탈 바이올렛으로 염색하였다. ACHN cell lines with lentivirus overexpressing RNF20 or SREBP1c were cultured in 6-well plates. Cells were incubated at 5% carbon dioxide at 37 ° C. for 7 days, then fixed with formaldehyde and stained with crystal violet.
세포주기 분석Cell cycle analysis
트립신에 처리된 세포를 인산완충식염수(PBS)로 세척한 후 4℃에서 70% 에탄올을 30분 처리하여 고정하였다. 고정된 세포를 인산완충식염수로 두 번 세척한 후 0.1% 노니뎃 P-40, 100μg/ml RNA 분해효소와 2.5μg/ml 프로피디움 아이오딘화합물(PI, propodium iodide)을 함유한 용액에 30분간 염색하였다. 염색된 세포를 FACS 칸토 II 기계를 이용한 유동세포분석법으로 분석했으며, 각 세포주기에 있는 세포 수는 ModFit LTTM세포주기 분석 프로그램을 통해 계산하였다.Trypsin-treated cells were washed with phosphate buffered saline (PBS) and fixed at 70 ° C. for 30 minutes at 4 ° C. The fixed cells were washed twice with phosphate buffered saline, and then rinsed in a solution containing 0.1% nonniche P-40, 100 μg / ml RNA degrading enzyme and 2.5 μg / ml propidium iodide (PI) for 30 minutes. Stained. Stained cells were analyzed by flow cytometry using the FACS Canto II machine, and the number of cells in each cell cycle was calculated using the ModFit LTTM cell cycle analysis program.
세포 내 트리글리세리드 측정Intracellular Triglyceride Measurement
세포 내 트리글리세리드는 기존에 보고한 대로 세포융해물을 비색분석법을 통해 측정하였으며, 세포 내 단백질 mg 당 지질 mg으로 표현하였다 (1). 간단히 설명하면, 세포융해물을 5% 트리톤 X-100을 이용하여 추출한 후 80℃ 수조에 담그고 얼음에 담그는 과정을 2번 반복하였다. 12,000 rpm에 5분 동안 원심 분리한 후, 상층액을 모아 Infinity 트리글리세리드 분석법을 이용하여 세포 내 트리글리세리드 양을 측정하였다. 측정값은 BCA 단백질 정량키트로 분석한 전체 단백질 양으로 보정하였다. Intracellular triglycerides were measured by colorimetric analysis as previously reported and expressed in mg of lipid per mg of protein in cells (1). Briefly, the cell lysate was extracted using 5% Triton X-100, and then immersed in an 80 ° C. water bath and immersed in ice twice. After centrifugation at 12,000 rpm for 5 minutes, the supernatant was collected and the amount of intracellular triglyceride was measured using Infinity triglyceride assay. The measured value was corrected by the total protein amount analyzed by the BCA protein quantitative kit.
보디피 염색Bodyfi dyeing
ACHN 신장암 세포주를 10μM 베툴린 유무 조건에서 24시간 배양하고 인산완충식염수로 두 번 세척한 후, 4% 파라포름알데히드로 10분 동안 고정하였다. 고정된 세포를 트윈-20를 함유한 인산완충식염수로 2번 세척한 후, 암실에서 1시간 동안 보디피 493/503이 결합된 플루오레세인 아이소싸이오시안산염(FITC, fluorescein isothiocyanate)으로 염색하였다. 표본은 4’,6-다이아미디노-2-페닐인돌(DAPI)을 함유한 벡타실드 용액으로 염색하였으며 Zeiss LSM 700 공초점 현미경을 이용하여 관측하였다. 모든 사진은 같은 조건에서 관찰하고 분석하였다.The ACHN kidney cancer cell line was incubated for 24 hours with or without 10 μM betulin, washed twice with phosphate buffered saline, and then fixed for 4 minutes with 4% paraformaldehyde. The immobilized cells were washed twice with phosphate buffered saline containing Tween-20 and stained with fluorescein isothiocyanate (FITC) bound to bodiphy 493/503 for 1 hour in the dark. . Samples were stained with a betashield solution containing 4 ′, 6-diamidino-2-phenylindole (DAPI) and observed using a Zeiss LSM 700 confocal microscope. All pictures were observed and analyzed under the same conditions.
PTTG1 리포터 및 루시퍼레이즈 분석PTTG1 Reporter and Luciferase Analysis
PTTG1 프로모터 부위(전사시작지점 -908 ~ +25 뉴클레오타이드)를 PCR하여 pGL3-basic 벡터에 클로닝하였다. 이전에 보고된 칼슘-인산 방법을 이용하여 DNA 플라스미드를 HEK293 세포주에 형질도입하였다 (4). 36시간 배양 후 형질도입된 세포를 수확하여 25 mM 트리스-인산(pH 7.8), 10% 글리세롤, 2 mM EDTA, 2 mM DTT, and 1% 트리톤 X-100이 함유된 버퍼를 사용하여 세포융해물을 추출하였다. 루시퍼레이즈 및 베타-갈라토시데이즈 활성은 제공사의 지시에 따라 측정하였다. 루시퍼레이즈 활성은 베타-갈라토시데이즈 활성으로 보정하였다.PTTG1 promoter region (transcription start point -908 to +25 nucleotides) was cloned into pGL3-basic vector. DNA plasmids were transduced into HEK293 cell line using the previously reported calcium-phosphate method (4). After 36 hours of incubation, the transduced cells were harvested and lysed using a buffer containing 25 mM tris-phosphate (pH 7.8), 10% glycerol, 2 mM EDTA, 2 mM DTT, and 1% Triton X-100. Was extracted. Luciferase and beta-galactidase activity were measured according to the instructions of the provider. Luciferase activity was corrected to beta-galactosidase activity.
이종 이식 실험(xenograft experiment)Xenograft experiment
피하 이종 이식 실험은 서울대학교병원의 동물실험관리원의 승인을 받았다(IACUC number: 13-0080). 1×107개의 대조군 ACHN 세포와 RNF20가 지속적으로 발현되는 ACHN 세포를 다섯 마리의 BALB/c 암컷 무흉선 누드 생쥐의 피하에 주입하였다. 주입 전 세포를 인산완충식염수 200μl에 풀고 동량의 마트리젤과 섞었다. 종양이 뚜렷이 나타난 후 종양 크기는 1주일마다 캘리퍼를 이용하여 측정하였으며 종양 부피는 다음의 공식으로 계산하였다. 부피(mm3)=(길이×너비2)×π/6. 이식 5주 후에 생쥐를 이산화탄소 흡입을 통해 안락사 시켰으며 이종 이식 종양을 꺼내 분석하였다. 이종 이식 조직 표본을 4% 파라포름알데히드에 고정시키고 30% 수크로스에 넣은 후 OCT 용액에 고정시켰다. 이종 이식 종양 조직 절편을 헤마톡실린/에오신(H/E)과 오일 레드 오 염색과 면역조직화학법을 수행하였다 (5). 간단히 설명하면 Ki67 단백질 및 TUNEL 기법은 스트렙타비딘-바이오틴 복합체 시스템을 이용하여 수행하였다. 사진은 EVOS ORIGINAL 현미경과 NIKON TMS 도립현미경을 이용하여 관찰하였다.Subcutaneous xenograft experiments were approved by the Animal Experimental Management Service of Seoul National University Hospital (IACUC number: 13-0080). 1 × 10 7 control ACHN cells and ACHN cells with continuous expression of RNF20 were injected subcutaneously in five BALB / c female athymic nude mice. Before injection, the cells were loosened in 200 μl of phosphate buffered saline and mixed with the same amount of Matrigel. After the tumor appeared clearly, the tumor size was measured using a caliper every week, and the tumor volume was calculated by the following formula. Volume (mm 3 ) = (length x width 2) x π / 6. Five weeks after transplantation, the mice were euthanized by inhalation of carbon dioxide and xenograft tumors were taken out and analyzed. Xenograft tissue samples were fixed in 4% paraformaldehyde and placed in 30% sucrose and then in OCT solution. Xenograft tumor tissue sections were subjected to hematoxylin / eosin (H / E) and oil red o staining and immunohistochemistry (5). Briefly, Ki67 protein and TUNEL techniques were performed using a streptavidin-biotin complex system. Photographs were observed using an EVOS ORIGINAL microscope and NIKON TMS inverted microscope.
통계 분석Statistical analysis
결과는 평균 ± 표준편차 또는 평균 ± 표준오차(그림 7B와 7C)로 표현되었다. 다군 비교는 일원분산분석 또는 두 가지 조건이 있을 경우 이원분산분석을 통해 수행하였다. 두 군의 차이는 양쪽 Student’s t-검정을 통해 분석하였다. 통계 분석은 Prism 그래프패드를 통해 수행하였으며 P < 0.05일 때 차이가 유의미하다고 보았다. Results were expressed as mean ± standard deviation or mean ± standard error (Figures 7B and 7C). Multigroup comparisons were performed by one-way ANOVA or two-way ANOVA if two conditions exist. Differences between the two groups were analyzed by both Student's t-tests. Statistical analysis was performed using the Prism graph pad and the difference was significant when P <0.05.
실시예 1. RNF20의 신장암 및 간암에서 하향조절 규명Example 1 Identification of Downregulation of Renal Cancer and Liver Cancer of RNF20
ccRCC(clear cell Renal Cancer Carcinmoa)에서는 이소성(ectopic) 지질 축적이 상당히 상향조절된다(Rezende et al., 1999, Differential diagnosis between monomorphic clear cell adenocarcinoma of salivary glands and renal (clear) cell carcinoma. Am J Surg Pathol 23, 1532-1538; Valera and Merino, 2011, Misdiagnosis of clear cell renal cell carcinoma. Nat Rev Urol 8, 321-333). RNF20이 SREBP1c를 저해함으로써 신생 지질 생성의 음성 조절자로 작용한다고 알려져 있으므로(Lee et al., 2014, Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation. Hepatology 60, 844-857), 본 발명자는 RNF20이 ccRCC 종양에서도 조절이상이 되는지를 조사하였다. 도 1의 A에 나타난 바와 같이, RNF20 mRNA 발현은 동일 환자 유래 정상 신장 조직과 비교하여 ccRCC 종양에서 상당히 하향조절되어 있다. 이와 유사하게, TCGA(the Cancer Genome Atlas)에서 얻은 RNA-Seq 데이타에서도 ccRCC 종양에서의 RNF20 mRNA 발현이 상당히 감소된 것으로 나타났는데, 이는 RNF20 발현이 낮은 것이 진행성 종양 단계와 밀접하게 연관된다는 것을 말한다(도 1C). 또한, 면역조직화학(IHC) 분석에서 RNF20 단백질 발현이 인접 정상 신장조직보다 ccRCC 종양에서 더 낮은 것으로 나타났다(도 1의 D). 이와 일치하게도, 동일 환자의 정상 신장 및 종양 조직에서 얻은 RNF20 염색 데이터에서도 ccRCC에서 RNF20 발현이 감소된 것으로 나타났다(도 1의 E). RNF20 발현은 또한, 인간 초기 신장 피질 내피세포(human primary renal cortical epithelial ; HRCE) 및 HEK293 정상 신장 세포에서와 비교하여 ccRCC 세포주 A498, Caki-2, 및 ACHN에서 감소되었다(도 9의 A). 또한 도 16에 나타난 바와 같이 RNF20은 단백질 및 유전자 수준에서 간암세포에서도 감소된 것으로 나타났다. Ectopic lipid accumulation is significantly upregulated in clear cell Renal Cancer Carcinmoa (Rezende et al., 1999, Differential diagnosis between monomorphic clear cell adenocarcinoma of salivary glands and renal (clear) cell carcinoma.Am J Surg Pathol) 23 , 1532-1538; Valera and Merino, 2011, Misdiagnosis of clear cell renal cell carcinoma.Nat Rev Urol 8 , 321-333). Since RNF20 is known to act as a negative regulator of angiogenesis by inhibiting SREBP1c (Lee et al., 2014, Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.Hepatology 60 , 844- 857) The inventors investigated whether RNF20 is dysregulated in ccRCC tumors. As shown in FIG. 1A, RNF20 mRNA expression is significantly downregulated in ccRCC tumors compared to normal kidney tissue from the same patient. Similarly, RNA-Seq data from the Cancer Genome Atlas (TCGA) showed a significant decrease in RNF20 mRNA expression in ccRCC tumors, indicating that low RNF20 expression is closely associated with advanced tumor stages ( 1C). In addition, immunohistochemistry (IHC) analysis showed that RNF20 protein expression was lower in ccRCC tumors than adjacent normal kidney tissues (FIG. 1D). Correspondingly, RNF20 staining data obtained from normal kidney and tumor tissues of the same patient also showed reduced RNF20 expression in ccRCC (FIG. 1E). RNF20 expression was also reduced in ccRCC cell lines A498, Caki-2, and ACHN as compared to human primary renal cortical epithelial (HRCE) and HEK293 normal kidney cells (FIG. 9A). As shown in FIG. 16, RNF20 was also reduced in liver cancer cells at the protein and gene levels.
RNF20 프로모터의 과메틸화에 의해 ccRCC에서 RNF20 발현이 하향조절되는지 알아보기 위하여, 본 발명자는 저해제인 RG108을 사용하여 DNA 메틸트랜스퍼라아제(DNMTs)를 저해하여 ccRCC 세포에서 RNF20 mRNA의 발현레벨을 측정하였다. RG108을 처리하였을 때, HEK293 비-암세포에는 영향을 미치지 않았고 ccRCC 세포에서 RNF20 발현을 상승시켰다(도 1의 F 및 9의 B). To determine if RNF20 expression is downregulated in ccRCC by hypermethylation of the RNF20 promoter, we measured the expression level of RNF20 mRNA in ccRCC cells by inhibiting DNA methyltransferases (DNMTs) using the inhibitor RG108. . Treatment with RG108 did not affect HEK293 non-cancer cells and elevated RNF20 expression in ccRCC cells (F and B in FIG. 1).
또한, 본 발명자는 RNF20 발현 및 임상결과 사이의 상관관계를 연구하여, RNF20의 발현이 낮은 것이 생존율이 낮은 것과 상당히 관련되어 있다는 것을 밝혔다(도 1G). RNF20과 낮은 생존율 사이의 이러한 밀접한 상관관계는 VHL 야생형 및 VHL 돌연변이 ccRCC 환자에서 관찰되었다(도 1H 및 1I). 따라서, 낮은 RNF20 발현에 의한 종양생성에 대한 결과를 평가하기 위하여, 본 발명자는 RNF20이 VHL 야생형 ACHN 및 VHL-결실된 A498 ccRCC 세포에서의 세포 증식에 영향을 미치는지를 알아보았다. 상기 실험에서, ACHN 및 A498 ccRCC 세포에서 RNF20의 과발현에 의해 세포증식이 억제되었다(도 1의 J 및 9C). 역으로, RNF20의 siRNA-매개 억제에 의해 ACHN 및 A498 세포를 포함한 ccRCC 세포주 및 간암세포주에서 세포 성장이 증가하였다(도 1K 및 9D, 도 20). 반대로, RNF20이 과발현되지 않거나 또는 siRNA-매개 녹다운되지 않으면, RNF20의 레벨이 높은 HRCE 및 HEK293 정상 신장 세포의 성장에 영항을 미치지 않았다 (도 9의 E-H). 상기 데이터는 RNF20이 VHL 상태와는 독립적으로 ccRCC 세포에서 종양 억제자로 작용할 수 있다는 것을 의미한다. In addition, we studied the correlation between RNF20 expression and clinical outcome, revealing that low expression of RNF20 is significantly associated with low survival (FIG. 1G). This close correlation between RNF20 and low survival was observed in VHL wild type and VHL mutant ccRCC patients (FIGS. 1H and 1I). Thus, to evaluate the results on tumorigenesis by low RNF20 expression, we examined whether RNF20 affects cell proliferation in VHL wild-type ACHN and VHL-deleted A498 ccRCC cells. In this experiment, cell proliferation was inhibited by overexpression of RNF20 in ACHN and A498 ccRCC cells (J and 9C in FIG. 1). Conversely, siRNA-mediated inhibition of RNF20 increased cell growth in ccRCC cell lines and liver cancer cell lines, including ACHN and A498 cells (FIGS. 1K and 9D, FIG. 20). In contrast, if RNF20 was not overexpressed or siRNA-mediated knocked down, the levels of RNF20 did not affect the growth of high HRCE and HEK293 normal kidney cells (FIG. 9E-H). The data means that RNF20 can act as a tumor suppressor in ccRCC cells independently of VHL status.
즉, 낮은 RNF20 발현이 VHL 돌연변이 상태와 상관없이 ccRCC 환자의 낮은 생존율과 강하게 연관되어 있었는데(도 1G-I), 이는 RNF20 발현이 ccRCC 진행과 역으로 관련되어 있다는 것을 의미한다. 이와 일치하게, RNF20의 이소성 발현이 VHL 야생형 및 결실된 ccRCC 세포주 둘다에서 SREBP1c 및 세포 증식을 억제하였지만(도 1J; 도 3A 및 F), 기저 RNF20 발현이 높은 HRCE 및 HEK293 정상 신장 세포의 증식에 영향을 미치지 않았다(도 9의 A 및 F).That is, low RNF20 expression was strongly associated with low survival of ccRCC patients regardless of VHL mutation status (FIG. 1G-I), which means that RNF20 expression is inversely related to ccRCC progression. Correspondingly, although ectopic expression of RNF20 inhibited SREBP1c and cell proliferation in both VHL wild type and deleted ccRCC cell lines (FIG. 1J; FIGS. 3A and F), basal RNF20 expression affected the proliferation of HRCE and HEK293 normal kidney cells with high expression. (A and F in FIG. 9).
따라서, 데메틸라제 저해제 RG108을 ACHN ccRCC 세포에 처리하면 RNF20 mRNF 발현이 실질적으로 증가하지만, HEK293 세포에서 관찰되지는 않았는데(도 9의 B), 이는 RNF20 프로모터 과메틸화가, 최소 일부분은, ccRCC에서 RNF20 발현을 감소시키게 할 수 있다는 것을 의미한다. Thus, treatment of demethylase inhibitor RG108 to ACHN ccRCC cells substantially increased RNF20 mRNF expression, but was not observed in HEK293 cells (FIG. 9B), which indicated that RNF20 promoter hypermethylation, at least in part, in ccRCC It is possible to reduce the expression of RNF20.
실시예 2. SREBP1 및 지방생합성 유전자의 신장암 및 간암에서의 상향조절 및 RNF20 발현과 역의 상관관계 규명Example 2. Inverse correlation of upregulation and RNF20 expression in renal and liver cancers of SREBP1 and adipose biosynthesis genes
교아세포종(glioma), 간암, 전립선암 및 췌장암과 같은 몇몇 타입의 종양에서 SREBP1 및 지방생합성 유전자가 고레벨로 발현되는데, 이는 또한 악성 진행 및 나쁜 결과와 양의 상관관계에 있다(Guo et al., 2009, EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy. Sci Signal 2, ra82; Huang et al., 2012, Activation of androgen receptor, lipogenesis, and oxidative stress converged by SREBP-1 is responsible for regulating growth and progression of prostate cancer cells. Mol Cancer Res 10, 133-142; Sun et al., 2015, SREBP1 regulates tumorigenesis and prognosis of pancreatic cancer through targeting lipid metabolism. Tumour Biol 36, 4133-4141). 그러나, SREBP1 및 지방생합성 유전자가 ccRCC에서 이소성 지질 저장과 연관되어 있는지는 불분명하다. 따라서 본 발명자는 정상 신장 및 ccRCC 종양 조직에서의 지방생합성 유전자의 발현 패턴을 분석하였다. 도 2A에 나타난 바와 같이, 동일 환자(patient-matched)의 정상 샘플과 비교하여 ccRCC 종양에서 SREBP1c mRNA가 상당히 상향조절되어 있었다. 또한, TCGA RNA-Seq 데이터에서, SREBP1이 ccRCC 종양에서 상향조절되어 있었고(도 2B), 진행성 종양 단계와 양의 상관관계(도 2C)인 것으로 나타나 있었다. 이러한 데이터와 일치하게도, FASN 및 SCD1에 대한 SREBP1 타겟 유전자의 mRNA 레벨이 ccRCC 종양에서 상승되었다(도 10A-F). 또한, SREBP1 및 지방생합성 효소인 FASN 및 SCD1의 단백질 발현이 동일 환자의 정상 신장 조직과 비교하여 ccRCC 종양에서 동시에 증가하였고, 반면 RNF20 단백질이 하향조절되었다(도 2D). 이러한 결과는 간암세포주를 사용한 실험에서도 관찰되었으며, 결과는 도 16에 기재되어 있다. In some types of tumors such as glioblastoma, liver cancer, prostate cancer and pancreatic cancer, SREBP1 and adipose biosynthesis genes are expressed at high levels, which is also positively correlated with malignant progression and bad outcomes (Guo et al., 2009, EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.Sci Signal 2, ra82; Huang et al., 2012, Activation of androgen receptor, lipogenesis, and oxidative stress converged by SREBP -1 is responsible for regulating growth and progression of prostate cancer cells.Mol Cancer Res 10 , 133-142; Sun et al., 2015, SREBP1 regulates tumorigenesis and prognosis of pancreatic cancer through targeting lipid metabolism.Tumor Biol 36 , 4133-4141 ). However, it is unclear whether SREBP1 and lipophilic genes are associated with ectopic lipid storage in ccRCC. Therefore, the present inventors analyzed the expression pattern of the adipose biosynthetic gene in normal kidney and ccRCC tumor tissue. As shown in FIG. 2A, SREBP1c mRNA was significantly upregulated in ccRCC tumors compared to normal samples of the same patient (patient-matched). In addition, the TCGA RNA-Seq data indicated that SREBP1 was upregulated in ccRCC tumors (FIG. 2B) and had a positive correlation with advanced tumor stages (FIG. 2C). Consistent with these data, mRNA levels of SREBP1 target genes for FASN and SCD1 were elevated in ccRCC tumors (FIGS. 10A-F). In addition, the protein expression of SREBP1 and the lipophilic enzymes FASN and SCD1 increased simultaneously in ccRCC tumors compared to normal kidney tissue of the same patient, while RNF20 protein was downregulated (FIG. 2D). These results were also observed in experiments using liver cancer cell lines, the results are described in FIG. 16.
다음으로, 본 발명자는 동일 ccRCC 환자에서 유래한 정상 신장 및 종양 조직 섹션을 IHV 염색을 하여 RNF20 및 SREBP1 발현 사이의 상관관계를 조사하였다. RNF20 시그널 강도는 ccRCC 종양 조직에서 감소하였지만, 반면 SREBP1 시그날을 증가하였다(도 2E). 이어 TCGA 분석으로 ccRCC 종양 조직에서 RNF20 및 SREBP1 발현이 서로 역 상관관계임을 밝혔다(도 2F). Next, we examined the correlation between RNF20 and SREBP1 expression by IHV staining of normal kidney and tumor tissue sections from the same ccRCC patient. RNF20 signal intensity decreased in ccRCC tumor tissue, while increasing SREBP1 signal (FIG. 2E). TCGA analysis then revealed an inverse correlation of RNF20 and SREBP1 expression in ccRCC tumor tissue (FIG. 2F).
결과적으로, RNF20의 mRNA 발현은 SREBP1c 타겟 유전자 FASN 및 ELOVL6의 것과 역으로 연관되어 있고(도 10G 및 H), FASN mRNA 발현은 낮은 생존율과 양으로 연관되어 있다(도 2G). 반대로, qRT-PCR 및 TCGA RNASeq 분석에서 SREBP2 및, 이의 타겟 유전자이며 콜레스테롤 합성의 속도제한 효소의 유전자인 HMGCR의 mRNA 레벨은 ccRCC 종양에서 감소되었다(도 10 I-L). As a result, mRNA expression of RNF20 is inversely correlated with that of the SREBP1c target genes FASN and ELOVL6 (FIGS. 10G and H), and FASN mRNA expression is positively associated with low survival (FIG. 2G). In contrast, mRNA levels of SREBP2 and its target gene, HMGCR, a gene that limits the rate of cholesterol synthesis, were reduced in ccRCC tumors in qRT-PCR and TCGA RNASeq assays (FIG. 10 I-L).
종합하면, 상기 결과는 ccRCC에서 SREBP1 레벨이 상승되고, 그 결과 지방생합성 활성화가 증가되고 임상 결과가 더 나빠진다는 것을 의미한다.Taken together, the results indicate that SREBP1 levels are elevated in ccRCC, resulting in increased lipogenesis and worsening clinical outcomes.
ccRCC에서 VHL의 손실에 기인한 HIF의 구성성분 활성화가 병인적 대사 변경을 야기한다는 것은 잘 알려져 있다. 그러나, 신장-특이적 VHL 결핍 마우스에서는 ccRCC-유사 대사 표현형이 나타나지 않았는데, 이는 ccRCC 종양 형성에 대한 추가적 매카니즘이 있음을 의미한다. 상기 실시예 1에서, RNF20 하향조절에 의해 SREBP1c가 활성화됨으로써 ccRCC 종양생성이 촉진되다는 것이 입증되었다. RNF20 발현이 정상 신장 조직과 비교하여 ccRCC 종양에서 감소하였고(도 1A-E) SREBP1 및 지방생합성 유전자 발현과 역의 상관관계에 있다(도 2A-F; 도 10A-H).It is well known that constitutive activation of HIF due to loss of VHL in ccRCC causes etiological metabolic alterations. However, no ccRCC-like metabolic phenotype was seen in kidney-specific VHL deficient mice, indicating that there is an additional mechanism for ccRCC tumor formation. In Example 1 above, it was demonstrated that activating SREBP1c by RNF20 downregulation promotes ccRCC tumorigenesis. RNF20 expression was reduced in ccRCC tumors as compared to normal kidney tissue (FIGS. 1A-E) and is inversely correlated with SREBP1 and lipophilic gene expression (FIGS. 2A-F; FIGS. 10A-H).
ccRCC에서 지질이 풍부하고 지방생합성이 증가한다는 종래 관찰들과 일치하게, 본원에서는 SREBP1 및 지방생합성 활성이 상향조절된 것으로 나타났다(도 2A-E; eh 10 A-F). 그러나, SREBP2 및 HMGCR과 같은 이의 타겟 유전자의 mRNA 레벨이 ccRCC 종양에서 감소하였는데(도 10I-L), 이는 SREBP1 및 신규 지방생합성이 초기 종양생성 역할을 할 수도 있다는 것을 의미한다.Consistent with previous observations of lipid-rich and increased fat biosynthesis in ccRCC, it has been shown herein that SREBP1 and adipose biosynthesis activity are upregulated (FIGS. 2A-E; eh 10 A-F). However, the mRNA levels of its target genes, such as SREBP2 and HMGCR, have decreased in ccRCC tumors (FIG. 10I -L), meaning that SREBP1 and new adipose biosynthesis may play an initial tumorigenic role.
실시예 3. RNF20에 의한 신장암 및 간암 세포에서 SREBP1c의 억제를 통한 지방생합성 및 세포 증식 감소효과 확인Example 3. Confirmation of lipophilic synthesis and cell proliferation reduction effect by inhibition of SREBP1c in kidney and liver cancer cells by RNF20
ccRCC 세포에서 RNF20 및/또는 SREBP1c 과별현의 효과를 조사하였다. VHL 야생형 ACHN ccRCC 세포에서, RNF20의 이소성 발현에 의해 내인성 및 이소성 핵 SREBP1 단백질이 둘다 분명히 억제되었다(도 3A). 그러나 siRNA를 사용하여 RNF20을 급성으로 억제시키면 SREBP1c mRNA 및 단백질 발현이 증가되었다(도 11A 및 B).The effect of RNF20 and / or SREBP1c overexpression on ccRCC cells was investigated. In VHL wild type ACHN ccRCC cells, both endogenous and ectopic nuclear SREBP1 proteins were clearly inhibited by ectopic expression of RNF20 (FIG. 3A). However, acute inhibition of RNF20 with siRNA increased SREBP1c mRNA and protein expression (FIGS. 11A and B).
이와 일치하는 것으로 SREBP1c 과발현되면 ACHN 세포에서 FASN, ACC1, SCD1, 및 ELOVL6를 포함하는 지방생합성 유전자의 mRNA 레벨이 증가되었다(도 3B). 반대로, RNF20은 대조군 및 SREBP1c-과발현하는 ACHN 세포 둘다에서 지방생합성 유전자의 mRNA 발현을 강력히 저해하였다(도 3B). 그러나 RNF20을 억제하면 지방생합성 유전자의 mRNA 발현을 증가시키고, 그리고 RNF20 및 SREBP1 둘다 낙다운하면 지방생합성 유전자 발현에 대한 RNF20 siRNA의 효과가 없어졌다(도 11C). 따라서, 세포내 트리글리세라이드 축적은 대조군 ACHN 세포에서보다 SREBP1c-과발현시키는 ACHN 세포에서 더 컸다(도 3C). 역으로, RNF20을 억제하면 세포내 트리글리세라이드 레벨이 증가하였다(도 11D). 상기와 같은 siRNF20을 이용한 RNF20의 억제는 간암세포주에서 SREBP1 단백질 농도를 증가시키고 지방생합성이 증가하는 것으로 나타났다 (도 21 및 도 22). Consistent with this, overexpression of SREBP1c resulted in increased mRNA levels of adipose biosynthesis genes including FASN, ACC1, SCD1, and ELOVL6 in ACHN cells (FIG. 3B). In contrast, RNF20 strongly inhibited the mRNA expression of adipose biosynthetic genes in both control and SREBP1c-overexpressing ACHN cells (FIG. 3B). However, inhibition of RNF20 increased the mRNA expression of adipose biosynthetic genes, and knocking down both RNF20 and SREBP1 eliminated the effect of RNF20 siRNA on adipose biosynthetic gene expression (FIG. 11C). Thus, intracellular triglyceride accumulation was greater in SREBP1c-overexpressing ACHN cells than in control ACHN cells (FIG. 3C). Conversely, inhibition of RNF20 increased intracellular triglyceride levels (FIG. 11D). Inhibition of RNF20 using siRNF20 as described above was found to increase the SREBP1 protein concentration and increase the fat biosynthesis in liver cancer cell lines (FIGS. 21 and 22).
SREBP1c 과발현이 ccRCC 세포에서 PCNA, cyclin A, D1, 및 E를 포함한 세포주기 조절자의 mRNA 발현을 촉진시켰으며(도 3D), 나아가 RNF20 과발현은 과발현된 SREBP1c의 효과를 감소시켰다(도 3D). RNF20 낙다운은 또한 ccRCC 세포에서 세포주기 유전자 발현을 촉진시켰다(도 11E). 반대로, RNF20 과발현은 콜로니 형성을 감소시킨 반면 이소성 SREBP1c 발현은 ACHN 세포에서 콜로니 형성을 증가시켰다(도 3E). 또한, SREBP1c 과발현은 ccRCC 세포 증식을 강화시켰으며(도 3F), SREBP1의 낙다운은 대조군 및 RNF20-억제 세포 둘다에서 ccRCC 세포 증식을 감소시켰다(도 11F). 상기와 같은 결과는 간암세포주에서도 관찰되었으며, 결과는 도 17, 18 및 19에 기재되어 있다. SREBP1c overexpression promoted mRNA expression of cell cycle regulators including PCNA, cyclin A, D1, and E in ccRCC cells (FIG. 3D), and further, RNF20 overexpression reduced the effect of overexpressed SREBP1c (FIG. 3D). RNF20 knockdown also promoted cell cycle gene expression in ccRCC cells (FIG. 11E). In contrast, RNF20 overexpression reduced colony formation whereas ectopic SREBP1c expression increased colony formation in ACHN cells (FIG. 3E). In addition, SREBP1c overexpression enhanced ccRCC cell proliferation (FIG. 3F) and knockdown of SREBP1 decreased ccRCC cell proliferation in both control and RNF20-inhibited cells (FIG. 11F). The same results were observed in liver cancer cell lines, and the results are described in FIGS. 17, 18 and 19.
이러한 데이터는 RNF20이 SREBP1c-유도 지방생합성 및 세포주기 진행을 억제시킴으로써 ccRCC 세포 증식을 저해한다는 것을 나타내는 것으로, RNF20이 신장암 치료제로 유용하게 사용될 수 있음을 나타내는 것이다. These data indicate that RNF20 inhibits ccRCC cell proliferation by inhibiting SREBP1c-induced fat biosynthesis and cell cycle progression, indicating that RNF20 can be usefully used as a therapeutic agent for kidney cancer.
또한 지방생합성을 억제하는 약물을 ccRCC에 대한 항암약물로 사용될 수 있음을 나타내는 것이다. 본원에서는 SREBP 저해제 베툴린이 VHL 유전자 돌연변이와 상관없이 SREBP1 및 지방생합성을 저해함으로써 ccRCC 세포 증식을 억제하는 것으로 나타났다(도 5A-C). 또한, 베툴린은 RNF20의 억제 후의 세포 증식 및 지방생합성 활성 증가를 제거하였다(도 13A-D). ccRCC에서 RNF20 하향조절과 더불어 SREBP1 및 지방생합성이 과활성화되면(도 1 및 2), 자유로운 SREBP1은 지방생합성 활성화를 통해 ccRCC 종양 발달을 촉진하는 것을 나타났다. 따라서, SREBP1 및 지방생합성 경로가 ccRCC에 대한 치료 타겟이 될 수 있음을 나타내는 것이다. It also indicates that drugs that inhibit fat biosynthesis can be used as anticancer drugs against ccRCC. It has been shown herein that the SREBP inhibitor betulin inhibits ccRCC cell proliferation by inhibiting SREBP1 and adipose biosynthesis irrespective of the VHL gene mutation (FIGS. 5A-C). In addition, betulin eliminated cell proliferation and increased lipogenic activity after inhibition of RNF20 (FIGS. 13A-D). When SREBP1 and lipogenesis were overactivated with RNF20 downregulation in ccRCC (FIGS. 1 and 2), free SREBP1 was shown to promote ccRCC tumor development through activating lipogenesis. Thus, it indicates that SREBP1 and adipose biosynthesis pathways can be therapeutic targets for ccRCC.
실시예 4. 신장암 세포에서 SREBP1c의 새로운 타겟 유전자로서 PTTG1 규명Example 4 Identification of PTTG1 as a New Target Gene of SREBP1c in Kidney Cancer Cells
전사 활성인자로서, SREBP1c는 지방산 대사 및 세포 주기 진행을 자극한다고 보고되어 있다(Bengoechea-Alonso and Ericsson, 2006, Cdk1/cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis. Cell Cycle 5, 1708-1718; Jeon et al., 2013, An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis. Cell Metab 18, 51-61; Williams et al., 2013, An essential requirement for the SCAP/SREBP signaling axis to protect cancer cells from lipotoxicity. Cancer Res 73, 2850-2862). ccRCC에서 SREBP1c-유도 세포 주기 진행에 관여하는 인자를 더 확인하기 위하여, 본 발명자는 야생형 및 SREBP1c 결핍 마우스의 간 조직에서 RNA-Seq 분석을 이용하여 세포주기 조절에 관여하는 SREBP1c 타겟 유전자를 분석한 결과, SREBP1c의 새로운 타겟 유전자로서 PTTG1을 확인하였다(도 4A).As transcriptional activators, SREBP1c has been reported to stimulate fatty acid metabolism and cell cycle progression (Bengoechea-Alonso and Ericsson, 2006, Cdk1 / cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis. Cell Cycle 5 , 1708-1718; Jeon et. al., 2013, An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis.Cell Metab 18, 51-61; Williams et al., 2013, An essential requirement for the SCAP / SREBP signaling axis to protect cancer cells from lipotoxicity.Cancer Res 73 , 2850-2862). To further identify factors involved in SREBP1c-induced cell cycle progression in ccRCC, the present inventors analyzed the SREBP1c target gene involved in cell cycle regulation using RNA-Seq analysis in liver tissues of wild-type and SREBP1c deficient mice. , PTTG1 was identified as a new target gene of SREBP1c (FIG. 4A).
도 12A에 나타난 바와 같이, PTTG1 발현은 야생형 마우스와 비교하여 SREBP1c 결핍 마우스의 신장, 간 및 지방조직에서 현저히 감소되어 있었다. 결과적으로, 본 발명자는 SREBP1c-과발현 ACHN 세포에서의 PTTG1 발현을 조사하였고 이소성 SREBP1c 발현이 PTTG1 mRNA 발현을 증가시키는 반면, RNF20 공동 발현이 이러한 효과를 약화시킨다는 것을 발견하였다(도 4B). 그러나, SREBP1 낙다운 조건하에서, RNF20은 PTTG1 mRNA (도 4C) 또는 단백질(도 4D) 발현을 억제하지 않았다. 따라서, SREBP1c는 ccRCC 세포에서 PTTG1 단백질 발현을 촉진시켰다(도 4E, 레인 3). 그러나, PTTG1의 siRNA 매개 억제는 SREBP1c 또는 FASN 단백질 발현에 영향을 미치지 않았다(도 4E, 레인 4). SREBP1c가 PTTG1전사를 직접적으로 조절하는지를 입증하기 위하여, 본 발명자는 인간, 원숭이, 개, 마우스 및 랫트의 PTTG1 유전자의 프록시말 프로모터 부위에 있는 SREBP1c에 대한 결합부위인, EBOX 모티프 및 잠정적 SRE(sterol regulatory element)를 분석하였다(도 12B). 그 결과 인간 PTTG1 프로모터 존재하의 루시퍼라아제 리포터 분석에서, SREBP1c 발현에 의해 루시퍼라아제 활성 및 RNF20의 공동발현이 억제된 SREBP1c-매개 PTTG1 프로모터 활성화가 유도되었다(도 4F). ccRCC 종양에서, 동일 환자 유래의 정상 신장조직에서와 비교하여, PTTG1 mRNA 레벨이 상당히 상향조절되었다(도 4G). 또한 TCGA 분석에서 PTTG1 mRNA 레벨이 ccRCC 종양조직에서 상당히 증가하였고(도 4H), PTTG1 mRNA 발현은 종양단계 진행과 양적으로 연관되어 있었다(도 4I). 또한, PTTG1 발현은 ccRCC 종양 조직에서의 RNF20 발현과 역의 상관관계에 있었다(도 4J). 더욱이, PTTG1가 고발현되는 것이 ccRCC 환자의 생존율이 낮은 것과 관련이 있었다(도 4K).As shown in FIG. 12A, PTTG1 expression was significantly reduced in kidney, liver and adipose tissue of SREBP1c deficient mice compared to wild type mice. As a result, we examined PTTG1 expression in SREBP1c-overexpressing ACHN cells and found that ectopic SREBP1c expression increased PTTG1 mRNA expression, while RNF20 co-expression attenuated this effect (FIG. 4B). However, under SREBP1 knockdown conditions, RNF20 did not inhibit PTTG1 mRNA (FIG. 4C) or protein (FIG. 4D) expression. Thus, SREBP1c promoted PTTG1 protein expression in ccRCC cells (FIG. 4E, lane 3). However, siRNA mediated inhibition of PTTG1 did not affect SREBP1c or FASN protein expression (FIG. 4E, lane 4). To demonstrate that SREBP1c directly modulates PTTG1 transcription, the inventors have identified an EBOX motif and a potential SER (binding regulatory site), a binding site for SREBP1c at the proximal promoter site of PTTG1 gene in humans, monkeys, dogs, mice and rats. element) was analyzed (FIG. 12B). As a result, in the luciferase reporter assay in the presence of the human PTTG1 promoter, SREBP1c expression induced SREBP1c-mediated PTTG1 promoter activation, which inhibited luciferase activity and co-expression of RNF20 (FIG. 4F). In ccRCC tumors, PTTG1 mRNA levels were significantly upregulated in comparison with normal kidney tissue from the same patient (FIG. 4G). In addition, PTTG1 mRNA levels were significantly increased in ccRCC tumor tissues in TCGA analysis (FIG. 4H), and PTTG1 mRNA expression was positively associated with tumor stage progression (FIG. 4I). In addition, PTTG1 expression was inversely correlated with RNF20 expression in ccRCC tumor tissue (FIG. 4J). Moreover, high expression of PTTG1 was associated with low survival of ccRCC patients (FIG. 4K).
종합하면, 상기 결과는 ccRCC에서 RNF20의 발현이 감소하고, 이는 활성화된 SREBP1c에 의해 새로운 타겟 유전자인 PTTG1의 고발현이 유도된다는 것을 나타낸다. Taken together, the results indicate that the expression of RNF20 in ccRCC decreases, indicating that high expression of PTTG1, a new target gene, is induced by activated SREBP1c.
상기에서 PTTG1이 또한 ccRCC에서 세포주기 진행 및 종양생성에 관여되어 있어 SREBP1c의 신규 타겟 유전자로 작용한다는 것을 보여주었다. 또한 PTTG1의 고 발현이 ccRCC환자에서 진행성 종양 단계 및 낮은 생존율과 밀접하게 연관되어 있다는 것을 밝혔다(도 4I 및 K). 또한, SREBP1은 PPTTG1의 mRNA 및 단백질 발현, 및 몇몇 세포-주기 조절자를 강력하게 자극시켜, ccRCC에서 암세포 증식이 일어났다(도 3 및 4). 이와 반대로 RNF20 과발현은 ccRCC 세포 및 이종이식 종양 둘다에서 PTTG1을 억제하며(도 4B; 도 7D 및 E), RNF20 억제에 의해 PTTG1의 mRNA 및 단백질 레벨이 증가하였다(도 4C 및 D).It has been shown that PTTG1 is also involved in cell cycle progression and tumorigenesis in ccRCC, acting as a novel target gene for SREBP1c. It was also found that high expression of PTTG1 is closely associated with advanced tumor stage and low survival in ccRCC patients (FIGS. 4I and K). In addition, SREBP1 strongly stimulated mRNA and protein expression of PPTTG1, and several cell-cycle regulators, resulting in cancer cell proliferation in ccRCC (FIGS. 3 and 4). In contrast, RNF20 overexpression inhibited PTTG1 in both ccRCC cells and xenograft tumors (FIG. 4B; FIGS. 7D and E), and mRNA and protein levels of PTTG1 were increased by RNF20 inhibition (FIGS. 4C and D).
실시예 5. SREBP 저해제인 베툴린에 의한 신장암 세포 증식 저해 확인Example 5. Confirmation of inhibition of renal cancer cell proliferation by betulin, an SREBP inhibitor
베툴린은 SREBP 단백질의 단백질분해 과정을 저해하여 지질-저하 효과를 나타내는 약물학적 저해제이다(Soyal et al., 2015, Targeting SREBPs for treatment of the metabolic syndrome. Trends Pharmacol Sci 36, 406-416; Tang et al., 2011, Inhibition of SREBP by a small molecule, betulin, improves hyperlipidemia and insulin resistance and reduces atherosclerotic plaques. Cell Metab 13, 44-56). 또한, 베툴린은 세포-주기 조절자를 포함한 다양한 종양 인자를 저해함으로써 다양한 암의 성장을 약화시킨다(Chintharlapalli et al., 2007, Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res 67, 2816-2823; Li et al., 2014, Betulin inhibits lung carcinoma proliferation through activation of AMPK signaling. Tumour Biol 35, 11153-11158). 이에 본 발명자는 SREBP 저해에 대한 베툴린의 효과를 조사하여 ccRCC 세포에서의 항-종양효과를 측정하였다. VHL 야생형 ACHN 및 VHL-고갈 A498 ccRCC 세포에 베툴린을 처리한 결과, 핵 SREBP1 단백질이 용량-의존적 방식으로 감소한 반면, SREBP1의 전구체 형태는 영향을 받지 않았는데(도 5A), 이는 베툴린이 SREBP1 단백질의 프로세싱을 억제한다는 것을 의미한다. 이와 유사하게, 베툴린-처리 ccRCC 세포에서 PTTG1과 싸이클린 B1 및 E를 포함한 세포주기 조절자의 단백질 레벨이 감소하였다(도 5A). Betulin is a pharmacological inhibitor that exhibits a lipid-lowering effect by inhibiting the proteolytic process of SREBP protein (Soyal et al., 2015, Targeting SREBPs for treatment of the metabolic syndrome.Trends Pharmacol Sci 36 , 406-416; Tang et al., 2011, Inhibition of SREBP by a small molecule, betulin, improves hyperlipidemia and insulin resistance and reduces atherosclerotic plaques.Cell Metab 13 , 44-56). Betulin also attenuates the growth of various cancers by inhibiting various tumor factors, including cell-cycle regulators (Chintharlapalli et al., 2007, Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res 67 , 2816-2823; Li et al., 2014, Betulin inhibits lung carcinoma proliferation through activation of AMPK signaling.Tumor Biol 35 , 11153-11158). The present inventors investigated the effect of betulin on SREBP inhibition to determine the anti-tumor effect on ccRCC cells. Betulin treatment of VHL wild-type ACHN and VHL-depleted A498 ccRCC cells resulted in a decrease in nuclear SREBP1 protein in a dose-dependent manner, whereas the precursor morphology of SREBP1 was not affected (FIG. 5A). Means to suppress processing. Similarly, protein levels of cell cycle regulators, including PTTG1 and cyclins B1 and E, were reduced in betulin-treated ccRCC cells (FIG. 5A).
또한, 베툴린을 처리하면 지방생합성 효소인 FASN 및 SCD1의 단백질 발현이 감소되었다(도 5A). 베툴린이 ccRCC 세포 성장을 억제하는지 조사하기 위하여, 본 발명자는 ACHN 및 A498 세포에서의 세포 증식에 대한 베툴린의 효과를 측정하여(도 5B 및 5C), 용량 의존적 항-증식 효과를 보여주었다. 이어, RNF20 억제 후 ccRCC 세포 증식 증가가 SREBP 활성화에 필요한지를 알아보기 위하여, ccRCC 세포에 베툴린 및 RNF20 siRNA를 처리하였다. 상기 결과와 일치하게도(도 1K), RNF20 siRNA 존재시에 ccRCC 세포 증식이 증가하였으며(도 13C 및 D), 이와 같은 조건에서 베툴린은 세포성장을 약화시켰다(도 13C 및 D). Treatment with betulin also reduced the protein expression of the fat biosynthetic enzymes FASN and SCD1 (FIG. 5A). To investigate if betulin inhibits ccRCC cell growth, we measured the effect of betulin on cell proliferation in ACHN and A498 cells (FIGS. 5B and 5C), showing a dose dependent anti-proliferative effect. Next, ccRCC cells were treated with betulin and RNF20 siRNA to see if increased ccRCC cell proliferation is required for SREBP activation after RNF20 inhibition. Consistent with the above results (FIG. 1K), ccRCC cell proliferation was increased in the presence of RNF20 siRNA (FIGS. 13C and D), and under these conditions betulin weakened cell growth (FIGS. 13C and D).
또한, ccRCC 세포에서 RNF20을 억제하면 지방생합성 및 세포주기 조절에 관여하는 여러 베툴린-민감성 유전자의 발현을 증강시켰다(도 13A 및 B). In addition, inhibition of RNF20 in ccRCC cells enhanced the expression of several betulin-sensitive genes involved in adipose biosynthesis and cell cycle regulation (FIGS. 13A and B).
이러한 데이터는 RNF20가 SREBP1c 제어를 매개함으로써 지방생합성 및 세포주기 조절 유전자 발현 조절을 통해 ccRCC 세포의 성장을 억제할 수 있다는 것을 의미한다.These data indicate that RNF20 can inhibit the growth of ccRCC cells through mediating SREBP1c control and regulating lipobiosynthesis and cell cycle regulatory gene expression.
이러한 유전자 발현 변화는 또한 베툴린이 처리된 ccRCC 세포에서 세포내 지질 축적이 감소하는 것과 일치한다(도 5D). PTTG1 및 특정 싸이클린을 포함한, 세포-주기 조절 유전자의 발현이 감소되는 것과 일치하게(도 5A), 베툴린은 약간 그러나 실질적으로 G1기에서 ccRCC 세포 수를 증가시킨다(도 5E). 이러한 데이터는 베툴린이 SREBP1-의존적 지방생합성 및 세포 주기 진행을 조절함으로써 ccRCC 세포 증식을 억제한다는 것을 의미한다. This gene expression change is also consistent with a decrease in intracellular lipid accumulation in betulin treated ccRCC cells (FIG. 5D). In line with the decrease in expression of cell-cycle regulatory genes, including PTTG1 and certain cyclins (FIG. 5A), betulin slightly but substantially increases the number of ccRCC cells at stage G1 (FIG. 5E). These data indicate that betulin inhibits ccRCC cell proliferation by regulating SREBP1-dependent lipobiosynthesis and cell cycle progression.
실시예 6. SREBP1c의 세포 주기 및 지방생합성에 영향을 미치는 이중 모드 작용을 통한 신장암 세포 성장 조절 규명Example 6 Identification of Renal Cancer Cell Growth Regulation Through Dual Mode Action Influencing Cell Cycle and Adipose Biosynthesis of SREBP1c
상기 실시예의 결과는 SREBP1c-의존적 ccRCC 세포 증식동안의 PTTG1 및 지방생합성사이의 상관관계를 나타내었다. 따라서, 본 발명자는 SREBP1c-과발현 ccRCC 세포에서 지방생합성 및/또는 세포 증식에 대한 PTTG1의 효과를 조사하였다(도 6A).The results of this example showed a correlation between PTTG1 and adipose biosynthesis during SREBP1c-dependent ccRCC cell proliferation. Thus, we investigated the effect of PTTG1 on lipobiosynthesis and / or cell proliferation in SREBP1c-overexpressing ccRCC cells (FIG. 6A).
PTTG1 억제는 SREBP1c 또는 FASN의 mRNA 발현을 변경시키지 않았지만(도 6B), 반면 이소성(ectopic) SREBP1c 발현은 ccRCC 세포에서 PCNA, cyclin A, D1, 및 E를 포함하는 세포-주기 조절자 및 PTTG1의 mRNA 발현을 촉진시켰다(도 6B 및 C). 반대로, ccRCC 세포에서 PTTG1 발현을 억제하면 이들 세포-주기단백질을 하향조절시켰다(도 6C). 또한, PTTG1을 억제하면 대조군 및 SREBP1c-과발현 ACHN 세포 둘다에서 세포 증식을 저해하였다(도 6D). PTTG1 inhibition did not alter the mRNA expression of SREBP1c or FASN (FIG. 6B), whereas ectopic SREBP1c expression did not affect cell-cycle regulators including PCNA, cyclin A, D1, and E in ccRCC cells and mRNA of PTTG1. Expression was promoted (FIGS. 6B and C). In contrast, inhibition of PTTG1 expression in ccRCC cells downregulated these cell-cycle proteins (FIG. 6C). In addition, inhibition of PTTG1 inhibited cell proliferation in both control and SREBP1c-overexpressing ACHN cells (FIG. 6D).
다음으로, 본 발명자는 ACC 저해제 TOFA 또는 FASN 저해제 C75 존재 또는 부존재시에 PTTG1 발현 및 세포 증식을 측정하였다(도 6A). 도 6E에 나타난 바와 같이 TOFA 및 C75를 처리하면 ACHN 세포에서 세포내 트리글리세라이드 축적이 감소되었다. 그러나 TOFA 또는 C75에 의한 지방생합성 활서의 감소는 PTTG1 또는 세포-주기 조절자 PCNA, 싸이클린 A, D1 및 E의 mRNA 발현에 상당한 영향을 미치지는 않았다(도 6F; 도 14A 및 B). 이와 유사하게, FASN의 siRNA-매개 억제가 PTTG1 및 세포-주기 조절 유전자의 mRNA 레벨을 크게 변경시키지는 않았다(도 14D 및 E). 그러나, FASN이 C75-매개 약물학적 저해되거나 siRNA-유도 FASN 낙다운된 경우 모두에서 ccRCC 세포 증식이 상당히 감소되었다(도 14C 및 F). 나아가, FASN의 약물학적 유전적 저해에 의해 ACHN 세포에서 세포 성장에 대한 SREBP1c 과발현의 효과를 감소시켰는데, 이는 지방생합성 저해가 SREBP1c-의존적 경로를 통해 ccRCC 세포 증식을 약화시킬 수 있다는 것을 의미한다(도 6G 및 F). Next, we measured PTTG1 expression and cell proliferation in the presence or absence of the ACC inhibitor TOFA or FASN inhibitor C75 (FIG. 6A). Treatment with TOFA and C75 as shown in FIG. 6E reduced intracellular triglyceride accumulation in ACHN cells. However, reduction of lipophilic activity by TOFA or C75 did not significantly affect the mRNA expression of PTTG1 or cell-cycle regulator PCNA, Cyclin A, D1 and E (FIG. 6F; FIGS. 14A and B). Similarly, siRNA-mediated inhibition of FASN did not significantly alter the mRNA levels of PTTG1 and cell-cycle regulatory genes (FIGS. 14D and E). However, ccRCC cell proliferation was significantly reduced in both cases where FASN was C75-mediated pharmacologically inhibited or siRNA-induced FASN knocked down (FIGS. 14C and F). Furthermore, the pharmacogenetic inhibition of FASN reduced the effect of SREBP1c overexpression on cell growth in ACHN cells, meaning that adipose biosynthesis inhibition could attenuate ccRCC cell proliferation via the SREBP1c-dependent pathway ( 6G and F).
종합하면 상기 결과는 SREBP1c-지방생합성 조절 기전과 더불어 새로이 규명한 SREBP1c-PTTG1 경로를 통해 세포주기를 조절함으로써 ccRCC 세포의 증식을 조절할 수 있다는 것을 나타낸다. Taken together, the results indicate that the proliferation of ccRCC cells can be regulated by regulating the cell cycle through the newly identified SREBP1c-PTTG1 pathway, along with the mechanism of SREBP1c-lipidogenesis.
상기 실시예 4에서 본 바와 같이, PTTG1 발현은 RNF20 발현과 역의 상관관계에 있어, ccRCC 종양 조직에서 SREBP1c의 조절을 반영하고(도 4J 및 8) RNF20 하향조절이, 일부분으로는, PTTG1을 상향조절시킴으로써 ccRCC 발달 및 진행을 촉진한다. 이와 일치하게도, PTTG1의 siRNA 낙다운은 지방생합성 활성을 변경시키기 않으면서 세포주기 조절 유전자의 mRNA 발현을 감소시키게 하였다(도 6C 및 E). 나아가, PTTG1 억제는 ccRCC 세포 증식에 대한 활성화된 SREBP1c의 효과를 약하게 하였다(도 6D). 따라서, 이러한 결과는 RNF20-SREBP1c-PTTG1 축이 ccRCC 세포 증식 및 종양생성의 중심이 됨을 나타낸다.As seen in Example 4 above, PTTG1 expression is inversely correlated with RNF20 expression, reflecting the regulation of SREBP1c in ccRCC tumor tissue (FIGS. 4J and 8) and RNF20 downregulation, in part, upwards PTTG1 Regulation promotes ccRCC development and progression. Correspondingly, siRNA knockdown of PTTG1 resulted in decreased mRNA expression of cell cycle regulatory genes without altering adibiosynthetic activity (FIGS. 6C and E). Furthermore, PTTG1 inhibition weakened the effect of activated SREBP1c on ccRCC cell proliferation (FIG. 6D). Thus, these results indicate that the RNF20-SREBP1c-PTTG1 axis is the center of ccRCC cell proliferation and tumorigenesis.
SREBP1이, 현저하게 신규 지방생합성을 증가시킴으로써 지방생합성를 조절한다는 것은 잘 알려져 있다. 따라서 본 발명자는 신규 지방생합성의 유전적 약물학적 저해제 존재시에 지방생합성이 PTTG1 발현과 연관되어 있는지를 시험하는데, ACHN ccRCC 세포에서 PTTG1의 mRNA 발현이 TOFA 또는 C75를 사용한 지방생합성의 약물학적 저해(도 6F) 또는 FASN의 siRNA 매개 낙다운(도 14D)에 의해 영향을 받지 않았다. 이는 SREBP1c에 의한 지방생합성 활성화 기전과 독립적으로 PTTG1이 유도된다는 것을 의미한다. 따라서, SREBP1c는 서로 다른 표적 유전자 군을 조절함으로써 지방생합성와 세포주기 진행에 영향을 미쳐, 궁극적으로 ccRCC에서 종양 발달을 촉진시킬 수 있다.It is well known that SREBP1 regulates fat biosynthesis by significantly increasing new fat biosynthesis. Therefore, the present inventors tested whether liposynthesis is associated with PTTG1 expression in the presence of novel lipophilic genetic pharmacological inhibitors, and the mRNA expression of PTTG1 in ACHN ccRCC cells was evaluated by pharmacological inhibition of lipobiosynthesis using TOFA or C75. 6F) or siRNA mediated knockdown of FASN (FIG. 14D). This means that PTTG1 is induced independent of the mechanism of lipophilic activation by SREBP1c. Thus, SREBP1c can influence different lipogenesis and cell cycle progression by regulating different target gene families, ultimately promoting tumor development in ccRCC.
실시예 7. RNF20 과발현에 의한 신장암 세포 이종이식에서 종양 성장 억제 효과 확인Example 7 Confirmation of Tumor Growth Inhibitory Effect in Renal Cancer Cell Xenograft by RNF20 Overexpression
인비보 ccRCC 종양 성장에 미치는 RNF20의 기능을 평가하기 위하여, 누드 마우스에서 암세포를 이식하여 이에 미치는 효과를 분석하였다. 그 결과 ACHN 이종이식 종양에서, 이소성 RNF20 발현에 의해 종양 성장 비율이 상당히 감소되었고(도 7A 및 B) 종양 질량 감소가 유도되었는데(도 7C), 이는 RNF20이 ccRCC 종양 성장을 억제할 수 있다는 것을 의미한다. 또한 웨스턴 블랏 분석에서 RNF20의 이소성 발현이 이종이식 종양에서 SREBP1, PTTG1 및 FASN의 단백질 발현을 저해하였다(도 7D). 또한, RNF20은 이종이식 종양에서 SREBP1c, 세포-주기 조절자, 및 지방생합성 유전자의 mRNA 발현을 약화시켰다(도 7E). H&E 염색실험에서, RNF20 발현이 높은 ACHN 종양은 투명한(clear) 세포 형태를 지닌 세포의 수가 감소된 것으로 나타났다(도 7F). 이와 일치하게도, 오일 레드 O 염색에서, 이소성 RNF20 발현에 의해 지질 축적이 감소되었다(도 7F). 또한 Ki67 염색 분석에서 외인성 RNF20 발현에 의해 이종이식 종양에서 세포 증식이 감소되었다(도 7F).To evaluate the function of RNF20 on in vivo ccRCC tumor growth, cancer cells were transplanted in nude mice and analyzed for their effects. As a result, in ACHN xenograft tumors, ectopic RNF20 expression significantly reduced tumor growth rates (FIGS. 7A and B) and induced tumor mass reduction (FIG. 7C), which means that RNF20 can inhibit ccRCC tumor growth. do. In addition, ectopic expression of RNF20 in Western blot analysis inhibited the protein expression of SREBP1, PTTG1 and FASN in xenograft tumors (FIG. 7D). In addition, RNF20 attenuated mRNA expression of SREBP1c, cell-cycle regulators, and lipophilic genes in xenograft tumors (FIG. 7E). In H & E staining, ACHN tumors with high RNF20 expression showed a reduced number of cells with clear cell morphology (FIG. 7F). Correspondingly, in oil red O staining, lipid accumulation was reduced by ectopic RNF20 expression (FIG. 7F). In addition, cell proliferation was reduced in xenograft tumors by exogenous RNF20 expression in Ki67 staining assay (FIG. 7F).
또한, TUNEL(terminal deoxynucleotidyl transferase dUTP nick end labeling ) 분석에서 RNF20 과발현에 의해 이종이식 종양에서 세포사멸이 유도되었다(도 7F). 또한 이소성 RNF20 발현 후에 Bax, Bid, 및 카스파제-3를 포함하는 향(pro)-세포사멸 유전자의 mRNA 발현이 증가되며, 반면 항-세포사멸 유전자 Bcl-2, cIAP-2, 및 XIAP의 항-세포사멸 유전자의 mRNA 발현은 감소한 것을 관찰하였다(도 15). 이러한 인비보 결과는, RNF20이 ccRCC에서 SREBP1c을 매개로 지방생합성 및 세포 주기 조절을 저해함으로써 종양억제자로 작용한다는 것을 의미한다. In addition, apoptosis was induced in xenograft tumors by RNF20 overexpression in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (FIG. 7F). In addition, mRNA expression of pro-apoptotic genes, including Bax, Bid, and caspase-3, is increased after ectopic RNF20 expression, whereas anti-apoptotic genes Bcl-2, cIAP-2, and XIAP -MRNA expression of apoptotic genes was observed to decrease (Fig. 15). These in vivo results indicate that RNF20 acts as a tumor suppressor by inhibiting SREBP1c-mediated adipose biosynthesis and cell cycle regulation in ccRCC.
종합하면 상기 결과는 RNF20의 과발현으로, SREBP1c 발현 및 지방생합성을 억제하여 암의 성장을 억제할 수 있음을 나타내는 것이다(도 7). 또한 상기 결과는 ccRCC에서 RNF20의 햐향 조절은 SREBP1c의 발현이 증가로 이어지고 이는 ccRCC에서 PTTG1 및 지방생합성 유전자의 발현이 증가로 이어져(도 8) 종양 성장 및 진행(progression)을 유발된다는 것을 의미한다. Taken together, the results indicate that overexpression of RNF20 can inhibit the growth of cancer by inhibiting SREBP1c expression and adipose biosynthesis (FIG. 7). The results also indicate that redirection of RNF20 in ccRCC leads to increased expression of SREBP1c, which in turn leads to increased expression of PTTG1 and lipophilic genes in ccRCC (FIG. 8), leading to tumor growth and progression.
또한 본원에서는 RNF20이 SREBP1c-매개 지방생합성 및 세포주기 조절을 저해함으로써 종양 억제자로 작용하는 모델을 제시하였다(도 8). 역으로, 상기 데이터는 RNF20 하향조절은 ccRCC 종양에서 SREBP1c를 활성화시킴으로써 종양생성을 촉진한다는 것을 의미하며, RNF20의 하향 발현은 암의 마커로 작용할 수 있다는 것이다. 또한 본 발명에서 SREBP1c가 ccRCC에서 PTTG1을 유도함으로써 세포주기 진행을 자극하는 새로운 매카니즘을 확인하였고, RNF20이 SREBP1c-지방생합성 축 및 SREBP1c-PTTG1 축을 조절할 수 있음을 밝혔다. Also presented herein is a model in which RNF20 acts as a tumor suppressor by inhibiting SREBP1c-mediated adipose biosynthesis and cell cycle regulation (FIG. 8). Conversely, the data indicate that RNF20 downregulation promotes tumorigenesis by activating SREBP1c in ccRCC tumors, and down expression of RNF20 may act as a marker of cancer. In the present invention, SREBP1c induced a PTTG1 in ccRCC to identify a new mechanism for stimulating cell cycle progression, and RNF20 can regulate the SREBP1c-lipid biosynthesis axis and the SREBP1c-PTTG1 axis.
이를 종합하여 보면, 본 발명에 의해 RNF20이 ccRCC에서 신규 종양 억제자이며 암에서 RNF20-SREBP1c-지방생합성 축 및 RNF20-SREBP1c-PTTG1 축 경로의 요소를 표적으로 하는 새로운 치료접근법이 임상적으로 효과가 있음을 나타내는 것이다. Taken together, the present invention demonstrates that the new therapeutic approaches that target RNF20-SREBP1c-lipid biosynthesis axis and RNF20-SREBP1c-PTTG1 axis pathways in cancer and RNF20 are novel tumor suppressors in ccRCC. It is present.
이상에서 본원의 예시적인 실시예에 대하여 상세하게 설명하였지만 본원의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본원의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본원의 권리범위에 속하는 것이다.Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다. All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Claims (18)

  1. RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 RNF20 유전자의 메틸화억제제를 포함하는, 신장암 또는 간암 예방 또는 치료용 약학 조성물. Ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a pharmaceutical composition for preventing or treating kidney cancer or liver cancer comprising a methylation inhibitor of the RNF20 gene.
  2. 제 1 항에서, 상기 신장암은 VHL (von Hippel-Lindau) 유전자 변이에 의한 것이 아닌, 신장암 또는 간암 예방 또는 치료용 약학 조성물. The pharmaceutical composition for preventing or treating kidney cancer or liver cancer of claim 1, wherein the kidney cancer is not caused by VHL (von Hippel-Lindau) gene mutation.
  3. RNF20(Ring finger protein 20) 검출용 물질을 포함하는, 신장암 또는 간암진단 또는 예후 측정용 조성물. Ring finger protein 20 (RNF20) comprising a substance for detecting, kidney cancer or liver cancer diagnostic or prognostic composition.
  4. 제 3 항에 있어서, The method of claim 3, wherein
    상기 검출용 물질은 상기 마커를 단백질 또는 핵산 수준에서 검출할 수 있는 시약으로, The detection material is a reagent that can detect the marker at the protein or nucleic acid level,
    상기 단백질 수준 검출 시약은 웨스턴블랏, ELISA, 방사선면역분석, 면역확산법, 면역 전기영동, 조직 면역염색, 면역침전 분석법, 보체 고정 분석법, FACS, 질량분석, 또는 단백질 마이크로어레이용 시약인, The protein level detection reagent is a reagent for Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray,
    상기 핵산 수준 검출 시약은 중합효소연쇄반응, 역전사 중합효소연쇄반응, 경쟁적 중합효소연쇄반응, Nuclease 보호 분석(RNase, S1 nuclease assay), in situ 교잡법, 핵산 마이크로어레이 또는 노던블랏에 사용되는 시약인, 신장암 또는 간암 진단 또는 예후 측정용 조성물.The nucleic acid level detection reagent is a reagent used in polymerase chain reaction, reverse transcriptase polymerase chain reaction, competitive polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray or Northern blot. , Kidney cancer or liver cancer diagnostic or prognostic composition.
  5. 제 4 항에 있어서, The method of claim 4, wherein
    상기 단백질 수준의 검출 시약은 상기 마커의 단백질 전장 또는 그 단편을 특이적으로 인식하는 항체, 항체단편, 앱타머(aptamer), 아비머(avidity multimer) 또는 펩티도모방체(peptidomimetics)를 포함하고, The protein level detection reagent comprises an antibody, antibody fragment, aptamer, avider or peptidomimetics that specifically recognizes the full length or fragment thereof of the marker,
    상기 핵산 수준의 검출 시약은 상기 마커의 핵산서열, 상기 핵산서열에 상보적인 핵산서열, 상기 핵산서열 및 상보적인 서열의 단편을 특이적으로 인식하는 프라미어 쌍, 또는 프로브, 또는 프라이머쌍 및 프로브인, 신장암 또는 간암 진단 또는 예후 측정용 조성물.The detection reagent at the nucleic acid level is a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair or probe, or a primer pair and a probe that specifically recognizes a fragment of the nucleic acid sequence and the complementary sequence. , Kidney cancer or liver cancer diagnostic or prognostic composition.
  6. RNF20-SREBP1c-PTTG1 신호전달 경로를 표적으로 하는 RNF20의 발현 이상과 관련된 암의 치료제 스크리닝 방법으로, 상기 방법은A method for screening a therapeutic agent for cancer associated with abnormal expression of RNF20 that targets the RNF20-SREBP1c-PTTG1 signaling pathway.
    RNF20의 발현이 감소 또는 결여된 세포를 제공하는 제 1 단계;Providing a cell with reduced or lacking expression of RNF20;
    상기 세포를 RNF20의 발현을 증가시킬 것으로 기대되는 시험물질로 처리하는 제 2 단계; A second step of treating said cells with a test substance that is expected to increase expression of RNF20;
    상기 시험물질로 처리된 세포에서 상기 SREBP1c 또는 PTTG1 중 하나 이상의 발현을 측정하는 제 3 단계; 및A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And
    상기 시험물질과 접촉된 세포와 상기 시험물질과 접촉되지 않은 대조군 세포에서 상기 SREBP1c 또는 PTTG1 발현을 비교하여, 상기 시험물질과 접촉된 세포에서 상기 SREBP1c 또는 PTTG1의 발현이 상기 대조군의 SREBP1c 또는 PTTG1 발현과 비교하여 감소한 경우, 이를 RNF20의 발현 이상과 관련된 암의 치료제 후보물질로 선별하는 제 4 단계를 포함하는, RNF20의 발현 이상과 관련된 암의 치료제 스크리닝 방법. Comparing the expression of the SREBP1c or PTTG1 in the cells in contact with the test substance and the control cells not in contact with the test substance, the expression of the SREBP1c or PTTG1 in the cells in contact with the test substance was compared with the expression of the SREBP1c or PTTG1 of the control. A method for screening a therapeutic agent for a cancer associated with abnormal expression of RNF20, comprising the fourth step of selecting, if reduced, a therapeutic agent candidate for cancer associated with abnormal expression of RNF20.
  7. 제 6 항에 있어서,The method of claim 6,
    상기 제 3 단계에서, 상기 SREBP1c의 발현을 측정하는 대신에, 또는 이에 부가하여, 상기 SREBP1c에 의해 발현이 촉진되는 지질생합성에 관여하는 유전자 또는 그 단백질의 발현을 측정하는 단계를 포함하며, In the third step, instead of or in addition to measuring expression of the SREBP1c, measuring the expression of a gene or a protein thereof involved in lipid biosynthesis promoted by the SREBP1c,
    이 경우 상기 제 4 단계에서 상기 후보물질은 상기 시험물질과 접촉된 세포에서 상기 시험물질과 접촉되지 않은 대조군 세포와 비교하여 상기 지질생합성에 관여하는 유전자 또는 단백질 발현이 감소한 것인, 방법.In this case, in the fourth step, the candidate is a decrease in the expression of the gene or protein involved in the lipid biosynthesis in the cells in contact with the test material compared to the control cells not in contact with the test material.
  8. 제 7 항에 있어서,The method of claim 7, wherein
    상기 지질생합성에 관여하는 단백질은 FASN, ACC1, SCD1, 또는 ELOVL6 중 하나 이상인, 방법.The protein involved in lipid biosynthesis is one or more of FASN, ACC1, SCD1, or ELOVL6.
  9. 제 6 항 또는 제 7 항에 있어서, The method according to claim 6 or 7,
    상기 RNF20의 발현이 감소 또는 결여된 세포는 신장암 또는 간암 세포주인, 방법.The cell having reduced or lacking expression of RNF20 is a kidney or liver cancer cell line.
  10. 제 6 항 또는 제 7 항에 있어서,The method according to claim 6 or 7,
    상기 RNF20의 발현 이상과 관련된 암은 신장암 또는 간암인, 방법.The cancer associated with abnormal expression of RNF20 is kidney cancer or liver cancer.
  11. 제 10 항에 있어서,The method of claim 10,
    상기 신장암은 VHL (von Hippel-Lindau) 유전자 변이에 의한 것이 아닌, 방법.The kidney cancer is not caused by VHL (von Hippel-Lindau) gene mutation.
  12. 신장암 또는 간암의 진단 또는 예후 판단을 위하여, For diagnosis or prognosis of kidney cancer or liver cancer,
    검사 대상체 유래의 생물학적 시료로부터 RNF20을 핵산 및/또는 단백질의 수준을 검출하는 단계;Detecting RNF20 levels of nucleic acids and / or proteins from a biological sample from a test subject;
    상기 핵산 및/또는 단백질 수준 검출결과를 대조군 시료의 해당 마커의 상응하는 결과와 비교하는 단계; 및Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And
    상기 대조군 시료와 비교하여, 상기 대상체 유래 시료의 핵산 또는 단백질 수준에 변화가 있는 경우, 상기 대상체를 신장암 또는 간암의 진단 또는 생존 예후와 연관시키는 단계를 포함하는, RNF20 바이오마커를 검출하는 방법.Comparing the control sample with a change in nucleic acid or protein levels in the subject-derived sample, associating the subject with a diagnosis or survival prognosis for kidney or liver cancer.
  13. 제 12 항에 있어서,The method of claim 12,
    상기 연관시키는 단계에서, 상기 대조군은 정상 대조군이며, 상기 대상체에서 결정된 RNF20의 수준이 상기 대조군과 비교하여 감소한 경우, 상기 대상제를 신장암 또는 생존 예후에서 5년 생존율이 낮은 것으로 판단하는 것을 포함하는 것인, 방법.In the associating step, the control group is a normal control group, and when the level of RNF20 determined in the subject is reduced compared to the control group, the subject agent includes determining that the 5-year survival rate is low in renal cancer or survival prognosis. How.
  14. RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현을 증가시키는 물질을 포함하는 인비트로 또는 인간을 제외한 동물에서 세포의 RNF20-SREBP1c-PTTG1 신호전달 경로 조절용 키트.Kit for regulating RNF20-SREBP1c-PTTG1 signaling pathway in cells in vitro or in animals other than humans, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene .
  15. RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현을 증가시키는 물질을 포함하는 인비트로 또는 인간을 제외한 동물세포의 RNF20-SREBP1c-지질생합성 경로 조절용 키트.Kit for adjusting the RNF20-SREBP1c-lipid biosynthesis pathway of animal cells, except in vitro or human, including a ring finger protein 20 (RNF20) gene, a protein encoded by the gene, or a substance that increases the expression of the gene.
  16. RNF20(Ring finger protein 20) 단백질 또는 이를 코딩하는 유전자의 신장암 또는 간암 진단용 용도. Ring finger protein 20 (RNF20) proteins or genes encoding them for the diagnosis of kidney or liver cancer.
  17. RNF20(Ring finger protein 20) 단백질 또는 이를 코딩하는 유전자의 인비트로 또는 인간을 제외한 동물세포의 RNF20-SREBP1c-지질생합성 경로 조절 용도. Ring finger protein 20 (RNF20) The in vitro use of the protein or gene encoding the same or for the regulation of the RNF20-SREBP1c-lipid biosynthesis pathway in animal cells other than humans.
  18. RNF20(Ring finger protein 20) 유전자, 또는 상기 유전자에 의해 코딩되는 단백질, 또는 상기 유전자의 발현을 증가시키는 물질을 포함하는 인비트로 또는 인간을 제외한 동물에서 세포의 RNF20-SREBP1c-PTTG1 신호전달 경로 조절 용도. For use in regulating RNF20-SREBP1c-PTTG1 signaling pathway in cells in vitro or in animals other than humans, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene .
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