WO2019088709A2 - Procédé de fourniture d'informations pour la prédiction d'un pronostic pour le cancer de l'ovaire utilisant le gène nc886 - Google Patents

Procédé de fourniture d'informations pour la prédiction d'un pronostic pour le cancer de l'ovaire utilisant le gène nc886 Download PDF

Info

Publication number
WO2019088709A2
WO2019088709A2 PCT/KR2018/013126 KR2018013126W WO2019088709A2 WO 2019088709 A2 WO2019088709 A2 WO 2019088709A2 KR 2018013126 W KR2018013126 W KR 2018013126W WO 2019088709 A2 WO2019088709 A2 WO 2019088709A2
Authority
WO
WIPO (PCT)
Prior art keywords
ovarian cancer
expression
gene
prognosis
tgf
Prior art date
Application number
PCT/KR2018/013126
Other languages
English (en)
Korean (ko)
Other versions
WO2019088709A3 (fr
Inventor
이용선
안지혜
이연수
최정혜
Original Assignee
국립암센터
경희대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 국립암센터, 경희대학교 산학협력단 filed Critical 국립암센터
Publication of WO2019088709A2 publication Critical patent/WO2019088709A2/fr
Publication of WO2019088709A3 publication Critical patent/WO2019088709A3/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a composition capable of diagnosing, preventing or treating ovarian cancer based on the expression level of nc886 gene.
  • the present invention also relates to a method for providing information for predicting the prognosis of ovarian cancer patients by confirming the expression level of ovarian cancer subtype and nc886 gene in ovarian cancer patients.
  • the information providing method includes an information providing method for identifying the expression level of the nc886 gene as an expression pattern of other genes related to nc886.
  • the present invention also relates to the use of a composition comprising an agent for measuring the level of expression of the nc886 gene for ovarian cancer prognosis prediction.
  • TGF- ⁇ Transforming growth factor- ⁇
  • miRNA microRNA pathway
  • TGF- ⁇ a cytokine that regulates a large number of target genes through SMAD transcription factors (TFs)
  • TFs SMAD transcription factors
  • TGF- ⁇ plays a role of carcinogenesis by promoting epithelial-mesenchymal transition (EMT) and fibrosis as malignant tumor progresses, although it is a tumor suppressor that suppresses cancer cell proliferation at the initial stage.
  • miRNAs non-coding RNAs (ncRNAs) that are processed by Dicer enzymes inhibit expression of messenger RNA (mRNA).
  • miRNAs such as miR-21, miR-155 and miR-17-92a-1 clusters are increased in some malignant tumors, but expression of most miRNAs is inhibited in cancer.
  • the overall down-regulation of miRNAs promotes tumorigenesis as evidenced by short hairpin RNA (shRNA) -mediated knockdown or improved tumor formation upon a single allele loss of Dicer.
  • shRNA short hairpin RNA
  • TGF- ⁇ and miRNA play an important role in the development, progression, and recurrence of ovarian cancer.
  • Most ovarian cancers are derived from the ovarian surface epithelium (OSE) and the tubal epithelium.
  • OSE ovarian surface epithelium
  • Epithelial tumors are composed of heterogeneous tumors and are classically classified into four major histological types (serous, mucinous, endometrioid, and clear cell).
  • the most common type of ovarian cancer is the high grade tubal epithelium, which represents a high frequency of TP53 mutations.
  • Mucinous, endometriosis, and low grade serous cancers are characterized by KRAS, ERBB2, BRAF, and PTEN mutations.
  • iM / fibrosis ovarian cancer subtypes
  • miRNA target genes are suppressed in other ovarian cancer subtypes called “integrated epithelial (iE) subtypes” or “oxidative stress” subtypes (hereinafter "iE / The reason why the TGF-beta and miRNA pathways are reversed according to the subtype of ovarian cancer is not studied or disclosed.
  • iE integrated epithelial subtypes
  • oxidative stress subtypes hereinafter “iE / The reason why the TGF-beta and miRNA pathways are reversed according to the subtype of ovarian cancer is not studied or disclosed.
  • nc886 is a long ncRNA of 101 nucleotides transcribed by RNA Polymerase III (Pol III).
  • Pol III RNA Polymerase III
  • An interesting feature of the nc886 genome region is the presence of strong CpG islands. Since Pol III activity is generally elevated in tumorigenesis, it is expected that nc886 levels will increase in cancer cells. However, nc886 has been shown to be sexually silent after some of the malignant tumors.
  • the present inventors confirmed that the expression of nc886 gene in ovarian cancer, particularly iM / fibrosis subtype, is increased, and the prognosis of ovarian cancer patients can be predicted according to the expression level of nc886. Respectively.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating ovarian cancer, which comprises an antisense oligonucleotide which inhibits the expression of nc886 gene.
  • Another object of the present invention is to provide a composition for diagnosing ovarian cancer of the iM / fibrosis subtype, which comprises an agent for measuring the expression level of the nc886 gene.
  • Yet another object of the present invention is to provide a method for diagnosing ovarian cancer, comprising the steps of: i) identifying ovarian cancer subtypes of ovarian cancer patients;
  • step iv) classifying the ovarian cancer patient of step ii) according to the expression pattern of nc886 identified in the step iii) into the nc886 highly positive group or the nc886 lowly expressed group;
  • the present invention provides a method for providing information for predicting the prognosis of a patient suffering from ovarian cancer.
  • Yet another object of the present invention is to provide a method for screening for gene expression data, comprising: i) obtaining gene expression data in A2780_vector, SKOV3_vector, OSE80PC_nc886-kd, SKOV3_nc886, OSE80PC_control-kd, A2780_nc886 and SKOV3_TGF-
  • step ii) analyzing the data obtained in step i) to obtain a pattern
  • step iii) integrating the pattern obtained in step ii) with a Bayesian compound covariate predictor (BCCP) algorithm to form a classifier;
  • BCCP Bayesian compound covariate predictor
  • step iv) classifying the gene expression data of patients with ovarian cancer into nc886 or nc886 low expression groups by the classifier formed in step iii);
  • the present invention provides a method for providing information for predicting the prognosis of a patient suffering from ovarian cancer.
  • Yet another object of the present invention is to provide a use for a composition for predicting ovarian cancer prognosis of the iM / fibrosis subtype of a composition comprising an agent for measuring the expression level of the nc886 gene.
  • the present invention can provide a pharmaceutical composition for preventing or treating ovarian cancer comprising an antisense oligonucleotide which inhibits the expression of nc886 gene.
  • the ovarian cancer may be an ovarian cancer of the iM / fibrosis subtype.
  • the present invention can also provide a composition for diagnosing ovarian cancer of the iM / fibrosis subtype comprising the agent for measuring the expression level of the nc886 gene.
  • the present invention also provides a method of treating ovarian cancer, comprising: i) obtaining a biological sample in an ovarian cancer patient;
  • step iii) classifying the ovarian cancer patient of step i) according to the expression pattern of nc886 identified in the step ii) into the nc886 highly positive group or the nc886 lowly expressed group;
  • the present invention provides a method for providing information for predicting the prognosis of a patient suffering from ovarian cancer.
  • the class of step iii) is ADAP2, ADM, ADM2, AHR, AK2P2, ALPP, ALS2CR14, ALS2CR4, ANXA2, ASS1, BCYRN1, C5orf13, C6orf170, C9orf130, CALD1, CCBE1, CCDC125 , CD68, CDKN1A, CENTA1, CEP27, CTGF, CYCSL1, CYR61, DDIT4, DENR, DHRS2, DOPEY2, DTWD2, EIF2AK4, ENO2, FAM153B, FAM40B, FAM72D, FAM73A, FLJ21986, FRMD6, FSTL1, GOS2, GDF15, GNAI1, GPT2 , HCFC1R1, HSCB, IER5L, IGFBP5, IL20RB, JAK1, KLHL28, LAMA5, LEP, MARCKS, MOAP1, NLRP8, NR4A2,
  • the information providing method comprises: iv) judging that the prognosis is poor when classified as the nc886 high incidence group as a result of the classification in the step iii); As shown in FIG.
  • the ovarian cancer may be an ovarian cancer of the iM / fibrosis subtype.
  • the poor prognosis may be indicative of cancer cell metastasis, resistance to chemotherapeutic agents, and death of the patient.
  • the anticancer agent may be paclitaxel.
  • the present invention provides a method for screening for gene expression, comprising: i) obtaining gene expression data in A2780_vector, SKOV3_vector, OSE80PC_nc886-kd, SKOV3_nc886, OSE80PC_control-kd, A2780_nc886 and SKOV3_TGF-?
  • step ii) analyzing the data obtained in step i) to obtain a pattern
  • step iii) integrating the pattern obtained in step ii) with a Bayesian compound covariate predictor (BCCP) algorithm to form a classifier;
  • BCCP Bayesian compound covariate predictor
  • step iv) classifying the gene expression data of patients with ovarian cancer into nc886 or nc886 low expression groups by the classifier formed in step iii);
  • the present invention provides a method for providing information for predicting the prognosis of a patient suffering from ovarian cancer.
  • the gene is selected from the group consisting of ADAP2, ADM, ADM2, AHR, AK2P2, ALPP, ALS2CR14, ALS2CR4, ANXA2, ASS1, BCYRN1, C5orf13, C6orf170, C9orf130, CALD1, CCBE1, CCDC125, CD68, CDKN1A , CENTA1, CEP27, CTGF, CYCSL1, CYR61, DDIT4, DENR, DHRS2, DOPEY2, DTWD2, EIF2AK4, ENO2, FAM153B, FAM40B, FAM72D, FAM73A, FLJ21986, FRMD6, FSTL1, GOS2, GDF15, GNAI1, GPT2, HCFC1R1, HSCB , IER5L, IGFBP5, IL20RB, JAK1, KLHL28, LAMA5, LEP, MARCKS, MOAP1, NLRP8, NR4A2, NU
  • the information providing method comprises the steps of: (v) predicting metastasis of ovarian cancer cells, resistance to chemotherapeutic agents and death of ovarian cancer patients when classified as nc886 high-risk group in step iv) Judging that it is bad;
  • the anticancer agent may be paclitaxel.
  • the present invention can provide a use for prediction of ovarian cancer prognosis of a composition comprising an agent for measuring the level of expression of nc886 gene.
  • the present invention can prevent or treat ovarian cancer by inhibiting the expression of nc886 gene, and can diagnose ovarian cancer by measuring the expression level of nc886 gene or predict the prognosis of ovarian cancer patients.
  • the present invention is effective as an information providing method for ovarian cancer prognosis prediction by analyzing the expression level of nc886 to predict the ovarian cancer metastasis or resistance to an anticancer agent.
  • FIG. 1 shows the expression levels of nc886 and TGFBI in ovarian cancer cell lines.
  • FIG. 2 shows the result of northern hybridization after treating SKOV3 and A2780 cells with 10M of 5-Aza-2'deoxycytidine (AzadC). It was confirmed that the expression of nc886 in each cell line was increased by treatment with AzadC.
  • FIG. 3 shows the scatter plot of nc886 and TGFBI expression values obtained from 25 patients with ovarian cancer at Cheil Hospital.
  • FIG. 4 shows the results of qRT-PCR analysis of the amount of TGFBI and SMAD5 expressed by Northern hybridization after the TGF-beta treatment of SKOV3 cells and the expression level of nc886. Expression of nc886 and TGFBI was significantly increased by TGF- ⁇ treatment, but SMAD5 expression was slightly increased. On the other hand, SKOV3 cells transfected to express DNA methyl-transferase (DNMT1) showed decreased expression of nc886 in spite of TGF- ⁇ treatment.
  • DNMT1 DNA methyl-transferase
  • nc886 RNA shows the genome region of nc886 of chromosome 5, the degree of methylation of the CpG site of nc886, and the heat map of EpiTYPER data.
  • the arrow indicates the transfer direction. All symbols in the magnified view (nc886 RNA, wavy lines, CpG islands, blue bars, EpiTYPER areas, dark magenta bars) were drawn at precise scales with nt coordinates computed based on the 5 'end of nc886 + The CpG positions (vertical bars) measured by EpiTYPER and pyrosequencing were designated dark magenta and purple, respectively.
  • FIG. 6 shows the quantification of nc886 expression levels in each cell line after northern hybridization. It was confirmed that the cell line transfected to express nc886 and the OSE80PC cell line stably expressed nc886.
  • 'SKOV3_TGF- ⁇ ' means SKOV3_ vector cells treated with TGF- ⁇ .
  • FIG. 7 shows the cell adhesion assay results of each cell line. Image, and quantification graph, and the graph shows the mean and standard deviation of pentaplicate. The expression of nc886 or TGF- ⁇ treatment increased cell adhesion.
  • FIG. 8 shows the results of cell migration analysis of each cell line. Image, and quantification graph, and the graph shows the mean and standard deviation of pentaplicate. The expression of nc886 or TGF- ⁇ treatment increased the cell migration capacity.
  • FIG. 9 shows the results of cell invasion analysis of each cell line. Image, and quantification graph, and the graph shows the mean and standard deviation of pentaplicate. The expression of nc886 or TGF- ⁇ treatment increased cell penetration ability.
  • FIG. 10 shows the migration ability of a cell line when SKOV3 cell line is treated with TGF-beta and / or nc886 kd.
  • TGF- ⁇ treatment increased the cell migration capacity, but knockdown (kd) of nc886 inhibited the effect.
  • Figure 11 represents the cell viability calculated as the MTT value, plotted against the concentration of paclitaxel.
  • Figure 12 shows the experimental plan for orthotopic implantation of ovarian cancer cells in nude mice and the number of mice showing ovarian cancer cell metastasis in each organ. When nc886 and TGF- ⁇ were treated, it was confirmed that ovarian cancer cells metastasized to other organs.
  • FIG. 13 shows a gene whose expression level is significantly changed according to ectopic expression of nc886 (“ nc886_exp ”) or TGF-beta treatment. Of these, 273 genes were identical, and the fc value was log2 scale.
  • FIG. 14 shows a scatter plot comparing fc values of 5221 genes modified by nc886 (x axis) and TGF-beta (y axis).
  • FIG. 15 shows nc886 and SNORD38B northern hybridization results as karyotype markers, and nc886 was mainly expressed in cytoplasm.
  • FIG. 16 is a scattergram comparing the fc value of 2,636 genes with the expression of nc886-kd (x axis) and nc886 (y axis).
  • FIG. 17 is a heat map showing unsupervised hierarchical clustering of genes associated with 1024 genes and 118 nc886 and expression levels. Selection criteria for each gene are shown on the right. The corresponding expression level, which is the array value associated with the median value of the sample, is displayed in green to red (see the color bar below for scales). Seven experiments that were performed three times in each experiment showed that nc886 expression levels by nc886-low and nc886-high (blue bars and red bars at the top, respectively) according to each manipulation (ectopic expression by nc886 kd and TGF- ⁇ treatment) Group. The blue / red color signature (nc886-low / -high) was used in all bar graphs and plots.
  • FIG. 18 shows the results of qRT-PCR of some genes whose expression level is related to nc886. The expression level of the genes was increased according to nc886 expression or TGF- ⁇ treatment.
  • the red data are the NF- ⁇ B-related pathways (IL1R_PATHWAY, NFKB_PATHWAY, NTHI_PATHWAY, CD40_PATHWAY), and the size marker's kilodalton (kD) is shown on the right.
  • IL1R_PATHWAY the NF- ⁇ B-related pathways
  • NFKB_PATHWAY NFKB_PATHWAY
  • NTHI_PATHWAY CD40_PATHWAY
  • kD size marker's kilodalton
  • FIG. 20 shows the rank distribution of MIR (top) and TFT (transcription factors target; bottom) at nc886-kd (left) and TGF-beta treatment (right).
  • FIG. 21 shows the scatter plot for the Z score of the MIR or TFT of nc886 kd and TGF- [beta] treatment on the left.
  • the top five candidate nc886-related miRNAs were selected and data points highlighted in red.
  • the heat map on the right shows clustering of genes that are highly modified in nc886 kd and TGF- ⁇ treatment.
  • One cluster contains 397 candidate miRNA target genes cross-compared with five miRNAs to directly target CNN, PDCD6 and ZEB2.
  • FIG. 22 shows a tactical miRNA qRT-PCR analysis including a normalized control group, small nuclear RNA U6 (U6 snRNA).
  • FIG. 23 shows a workflow for identifying miRNAs and target genes related to nc886 / TGF-beta in ovarian cancer.
  • Figure 25 shows cleaved mutations with the nc886-related top 12 proteins and Dicer domains determined through mass spectrometry.
  • FIG. 26 shows Western blotting results and nc886-binding assay results.
  • FIG. 27 shows the results of in vitro treatment and analysis of nc886 and pre-miR-200c together with an appropriate amount of FLAG-Dicer (WT) and FLAG-Dicer purified by FLAG IP. Maturation of the pre-miRNAs shown was visualized by Northern hybridization. Each bar in the graph corresponds to the corresponding band. Pre-miRNAs, mature miRNA and degradation products were quantified and plotted.
  • Figure 29 shows the expression of miR-mimic (miR-124-3p, -183-5p, -203a-3p, -200c-3p and -19b-3p) transfected into OSE80PC cells 24 hours after cell harvesting for RNA preparation. 3p) or qRT-PCR results of four genes due to non-microbial transfection. MRNA expression of the three genes (CNN3, PDCD6, ZEB2) that are not PRKCA is inhibited by miRNA mimic, indicating that PRKCA is not regulated by these miRNAs. Represents the 3'-untranslated region of the candidate miRNA target gene. This region was cloned into a plasmid downstream of the firefly luciferase (Pp) open reading frame.
  • Pp firefly luciferase
  • Luciferase was tested for transfection with miRNA-mimic or control-mimic with the plasmid. Analysis was performed 24 hours after transfection of the luciferase plasmid. Relative luciferase values (y-axis) were calculated from several standardizations. First, the value from Pp was normalized to the Renilla luciferase (Rr) value from co-transfected pRL-SV40. In each plasmid, the Pp / Rr value of the negative control mimic was set to one. Mean and standard deviation were calculated from three samples.
  • FIG. 30 shows the results of miRNA qRT-PCR, representative images and quantification graphs as a result of western blotting analysis of cell adhesion according to Dicer-kd for 24 hours.
  • FIG. 31 shows cell migration analysis according to ectopic expression of Dicer.
  • Western blot results for anti-FLAG antibodies show representative images and quantification plots.
  • FIG. 33 shows a schematic diagram of the parameters of the prediction model.
  • BCCP Bayesian Compound Covariate Predictor
  • LOOCV leave-one-out cross-validation.
  • the cutoff for Bayesian probability is nc886 high> 0.7 or nc886 low ⁇ 0.3.
  • FIG. 34 shows Kaplan-Meier plots for overall survival (OS) and recurrence-free survival (RFS). A total of 285 patients were stratified into two groups as predicted by the BCCP algorithm. The P value was generated by the log-rank test, and the + sign represents the censored data.
  • antisense of the present invention is also referred to as an antisense oligomer, and includes a sequence of a nucleotide base capable of hybridizing with a target sequence in RNA by Watson-Crick base pair formation to form mRNA and RNA: oligomer heterodimers in the target sequence, Means an oligomer having a backbone between subunits.
  • the antisense can have an exact sequence complement or approximate complement to the target sequence, block or inhibit the translation of the mRNA, and alter the processing of the mRNA producing splice variants of the mRNA.
  • the antisense of the present invention may preferably be an antisense oligomer complementary to the polynucleotide of the nc886 gene of the present invention.
  • the antisense can be used in a manner that prevents or suppresses the expression of a carcinogen gene by administration to a subject in a conventional manner.
  • a method of mixing an antisense oligodeoxynucleotide with a poly-L-lysine derivative by electrostatic attraction and administering the mixture to a vein of a subject (JS Kim et al., J controlled Release 53, 175-182 , 1998) may be used but are not particularly limited thereto.
  • prognosis of the present invention means a prognosis for a medical cause (e.g., long-term viability, disease-free survival rate, etc.) and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis) (Eg, recurrence, tumor growth, metastasis, drug resistance, etc.), positive prognosis may include disease progression such as a disease-free state, improvement or stabilization of disease such as tumor degeneration, .
  • a medical cause e.g., long-term viability, disease-free survival rate, etc.
  • positive prognosis may include disease progression such as a disease-free state, improvement or stabilization of disease such as tumor degeneration, .
  • the poor prognosis of ovarian cancer patients in the present invention indicates that metastasis of ovarian cancer cells occurs (Example 3, [ Figure 12]), resistance to ovarian cancer is present and ovarian cancer patients may die (Example 9, [ Figure 34] and [ Figure 35]).
  • prediction means a presumption of medical judgment, and it is an object of the present invention to provide a method for diagnosing ovarian cancer in a patient diagnosed with ovarian cancer (disease progression, improvement, Drug resistance) in advance.
  • agent for measuring the expression level of a gene means a molecule that can be used for confirming the expression level of the biomarker gene of the present invention, preferably a primer pair specifically binding to the gene, Probes or antisense nucleotides.
  • nc886 gene according to the present invention is induced by TGF-beta in ovarian cancer, particularly iM / fibrosis subtype, it can prevent or treat ovarian cancer using miRNA pathway, and its expression level The diagnosis of ovarian cancer or its prognosis can be predicted.
  • the present invention relates to a pharmaceutical composition for preventing or treating ovarian cancer comprising an antisense oligonucleotide inhibiting the expression of the nc886 gene or a pharmaceutical composition for inhibiting the expression of the iM / fibrosis subtype comprising the agent for measuring the expression level of the nc886 gene
  • a pharmaceutical composition for diagnosing ovarian cancer comprising an antisense oligonucleotide inhibiting the expression of the nc886 gene or a pharmaceutical composition for inhibiting the expression of the iM / fibrosis subtype comprising the agent for measuring the expression level of the nc886 gene
  • the ovarian cancer cell line especially the iM / fibrosis subtype ovarian cancer cell line, has a high expression level of nc886 gene, and when the expression level of nc886 gene is high, it can be predicted that the prognosis of ovarian cancer patient is bad.
  • the ovarian cancer of the iM / fibrosis subtype is an ovarian cancer which strongly expresses the mesenchymal-associated miRNAs (Prior Art 2), miR-200a and negative (ovarian cancer).
  • the substance that inhibits the expression of the nc886 gene may be an antisense oligonucleotide, an aptamer, an siRNA, or a shRNA specifically binding to the nc886 gene, but is preferably an antisense oligonucleotide.
  • the sequence may be 3'-UAGAGACACGACCCCAAGCU-5 ', and the sequence of the nc886 gene is the same as that of SEQ ID NO: 1.
  • the present invention also provides a method of treating ovarian cancer, comprising: i) obtaining a biological sample in an ovarian cancer patient;
  • step iii) classifying the ovarian cancer patient of step i) according to the expression level of nc886 identified in the step ii) into the nc886 highly immunized group or the nc886 low expressed group;
  • the present invention provides a method for providing information for predicting the prognosis of ovarian cancer patients.
  • the "biological sample” obtained from the ovarian cancer patient may be an ovarian-derived tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine of an ovarian cancer patient, preferably ovarian cancer tumor tissue.
  • classification into the nc886 high-incidence group or the nc886 low-expression group can be classified based on the expression pattern of the genes whose expression levels vary according to the expression of nc886.
  • the expression pattern of a total of 1024 genes confirmed in the present invention can be used, but more specifically, the expression pattern of the following 118 genes can be analyzed and used as a classification standard; ADL2, ADM2, AHR, AK2P2, ALPP, ALS2CR14, ALS2CR4, ANXA2, ASS1, BCYRN1, C5orf13, C6orf170, C9orf130, CALD1, CCBE1, CCDC125, CD68, CDKN1A, CENTA1, CEP27, CTGF, CYCSL1, CYR61, IGFBP5, IL20RB, JAK1, KLHL28, LAMA5, IGFBP5, IGFBP5, IL20RB, DENR, DHRS2, DOPEY2, DTWD2, EIF2AK4, ENO2, FAM153B, FAM40B, FAM72D, FAM73A, FLJ21986, FRMD6, FSTL1, G0S2, GDF15, GNAI1, GPT2, LIP, MARC
  • the ovarian cancer of the present invention is preferably an ovarian cancer of the iM / fibrosis subtype, but is not limited thereto.
  • the anticancer agent of the present invention is preferably paclitaxel, but it is not limited as long as it is an anticancer agent capable of treating ovarian cancer.
  • the present invention also provides a method for screening for gene expression, comprising: i) obtaining gene expression data in A2780_vector, SKOV3_vector, OSE80PC_nc886-kd, SKOV3_nc886, OSE80PC_control-kd, A2780_nc886 and SKOV3_TGF-?
  • step ii) analyzing the data obtained in step i) to obtain a pattern
  • step iii) integrating the pattern obtained in step ii) with a Bayesian compound covariate predictor (BCCP) algorithm to form a classifier;
  • BCCP Bayesian compound covariate predictor
  • step iv) classifying the gene expression data of patients with ovarian cancer into nc886 or nc886 low expression groups by the classifier formed in step iii);
  • the present invention provides a method for providing information for predicting the prognosis of ovarian cancer patients.
  • the A2780_vector, SKOV3_vector, and OSE80PC_nc886-kd cell lines are low cell line expressions of nc886, and SKOV3_nc886, OSE80PC_control-kd, A2780_nc886 and SKOV3_TGF- ⁇ cell lines are high cell line expressions of nc886 (FIG.
  • the patterns of the genes expressed in these cells are analyzed to obtain data which can serve as basis for judging whether the expression level of nc886 is high or not.
  • Gene expression data can be generated using the Affymetrix microarray platform (U133 v2.0), but can be used as a tool commonly used in the art to generate gene expression data.
  • the generated gene expression data can be analyzed using BRB-ArrayTools, but it can be used as a means commonly used in the art to generate gene expression data.
  • the pattern of the genes may be integrated into a BCCP (Bayesian compound covariate predictor) algorithm to form a classifier as a criterion for determining whether the expression level of nc886 is high.
  • BCCP Bayesian compound covariate predictor
  • the BCCP algorithm can determine whether the sample belongs to a specific subgroup, p ⁇ 0.05 for three experiments, and fold change> 1.3.
  • it can be used as a means commonly used in the art to analyze patterns of genes.
  • the degree of expression of nc886 in a specific ovarian cancer patient and further the prognosis of the patient can be predicted (Example 9).
  • classification into the nc886 high expression group or the nc886 low expression group can be classified based on the expression pattern of genes whose expression levels vary according to the expression of nc886.
  • the expression pattern of a total of 1024 genes confirmed in the present invention can be used, but more specifically, the expression pattern of the following 118 genes can be analyzed and used as a classification standard; ADL2, ADM2, AHR, AK2P2, ALPP, ALS2CR14, ALS2CR4, ANXA2, ASS1, BCYRN1, C5orf13, C6orf170, C9orf130, CALD1, CCBE1, CCDC125, CD68, CDKN1A, CENTA1, CEP27, CTGF, CYCSL1, CYR61, IGFBP5, IL20RB, JAK1, KLHL28, LAMA5, IGFBP5, IGFBP5, IL20RB, DENR, DHRS2, DOPEY2, DTWD2, EIF
  • the above information providing method of the present invention is characterized in that, when vcn86 is classified as a high-risk group in step iv), the prognosis of the ovarian cancer cell is judged to be poor by predicting metastasis of cancer cells, resistance to chemotherapy, and death of ovarian cancer patients step; As shown in FIG.
  • the anticancer agent of the present invention is preferably paclitaxel, but it is not limited as long as it is an anticancer agent capable of treating ovarian cancer.
  • the present invention also provides a use for predicting ovarian cancer prognosis of a composition comprising an agent that measures the level of expression of the nc886 gene. Since the nc886 gene and the agent for measuring the expression level are the same as those used in the above composition or information providing method, description thereof will be replaced by the description above. nc886 expression level is measured and it is predicted that the prognosis of the ovarian cancer patient is poor when it is separated into the nc886 high incidence group. The poor prognosis is because ovarian cancer cells are metastasized, resistant to anticancer drugs such as paclitaxel, Of the population can die.
  • the expression level of the nc886 gene can be directly measured, and the expression pattern of the next 118 genes related to the expression of the nc886 gene can be analyzed and used as a classification standard; ADL2, ADM2, AHR, AK2P2, ALPP, ALS2CR14, ALS2CR4, ANXA2, ASS1, BCYRN1, C5orf13, C6orf170, C9orf130, CALD1, CCBE1, CCDC125, CD68, CDKN1A, CENTA1, CEP27, CTGF, CYCSL1, CYR61, IGFBP5, IL20RB, JAK1, KLHL28, LAMA5, IGFBP5, IGFBP5, IL20RB, DENR, DHRS2, DOPEY2, DTWD2, EIF2AK4, ENO2, FAM153B, FAM40B, FAM72D, FAM73A, FLJ21986, FRMD6, FSTL1, G0S2, GDF15, GNAI1, GPT2,
  • SKOV3- and A2780-derived cell lines stably expressing nc886 were prepared as follows: Briefly, cells were transfected with nc886 expression plasmids (Table 3), treated with antibiotics (depending on the plasmid, puromycin, Or G418), single colonies were isolated and nc886 expression was confirmed by Northern hybridization.
  • Table 2 shows stable cell lines prepared and used in the present invention, and [Table 3] and [Table 4] show information of plasmids used in the production of the cell lines of Table 2 above.
  • TGF-beta (R & D Systems, Minneapolis, MN) 10 ng / ml was treated for 96 hours. Specifically, fresh TGF- ⁇ was supplemented daily by changing the culture medium for 96 hours, unless otherwise specified.
  • the source of the antibody and reagent used in the present invention is the same as that of the prior art 4 unless otherwise specified.
  • Stable cell line Parent cell line The plasmid used Remarks SKOV3_vector SKOV3 pLKO.1 - TRC Used for functional and mRNA analysis SKOV3_nc886 SKOV3 pLKO.1-886 (102T) A2780_vector A2780 pCAGGS-GFP A2780_nc886 A2780 pCAGGS-GFP / 886 SKOV3-GFP (vector) SKOV3 pCAGGS-GFP Used for functional analysis SKOV3-GFP (nc886) SKOV3 pCAGGS-GFP / 886 A2780-pLKO (vector) A2780 pLKO.1 - TRC A2780-pLKO (nc886) A2780 pLKO.1-886 (102T)
  • RNA concentration and purity were determined using NanoDrop ND-1000 (NanoDrop, Wilmington, DE) prior to qRT-PCR.
  • PCR primers were designed using EpiDesigner (Sequenom, http://www.epidesigner.com) and the sequence information is summarized in Table 4 below.
  • the PCR conditions for EpiTPYER are as follows; 15 minutes at 94 ⁇ (initial denaturation); 45 cycles of 94 ° C for 20 seconds, 56 to 62 ° C for 30 seconds, and 72 ° C for 60 seconds (amplification); 72 [deg.] C for 3 min (final extension).
  • EpiTYPER was performed using EpiTYPER Reagent and SpectroCHIP Set (Agena Bioscience, San Diego, Calif.) And data were analyzed using EpiTYPERTM ver 1.2 (Agena Bioscience) heat map).
  • mRNA analysis was performed using the TotalPrep TM RNA amplification kit and the HumanHT-12 v4.0 Expression BeadChip kit (Illumina, San Diego, Calif.). The degree of correlation is expressed as the Pearson's r value in scatter plots for the array fc values of pairs of samples and the significance of the linear regression model is shown in the F-statistics using R software (version 2.6.1) Based p-value. ≪ / RTI >
  • plasmid DNA in this study is summarized in Table 3 above.
  • PCR-amplified DNA fragments from genomic DNA were inserted into the indicated vector.
  • PCR was performed with the amfiFusion High Fidelity PCR Master Mix (GenDepot, Barker, TX) using the primers listed in Table 4 above.
  • siDicer and siPKR are Invitrogen's Stealth RNAi TM siRNA.
  • siRNA sequences are available upon request. siRNA was transfected at 40 nM for various times as indicated in the figure legend.
  • anti-nc886 targeted and non-targeted control antialligos designated as "anti-nc886" and "anti-control" in this study are 3'-UAGAGACACGACCCCAAGCU-5 'and 3'-CCGACCGAAATCGAGUCGCC-5', respectively.
  • Anti- oligo was transfected at 100 nM.
  • RNA double stranded miRNA mimic of ST Pharm (Siheung, Korea) was transfected for 24 h at 10 nM.
  • Small RNAs small RNAs (siRNA, anti-oligos and miRNA mimetics) were transfected with Lipofectamine TM RNAiMAX reagent (Invitrogen).
  • Lipofectamine TM 2000 reagent (Invitrogen) was used when plasmid DNA (alone or in combination with small RNA) was transfected.
  • MeT5A cells from a human mesothelial cell line (American Type Culture Collection, Manassas, VA) were plated on flat 96-well plates (10 6 cells per well) and allowed to adhere overnight to form a mesocarp layer. After experimental manipulations (e. G.
  • ovarian cells were isolated by trypsinization and washed with phosphate buffered saline (PBS) and probed with 10 [mu] M CellTraker (Invitrogen) for 45 min at 37 [ CellTraker TM - labeled cells were washed with 0.1% fetal bovine serum (FBS) and RPMI 1640 medium to remove free dye and stained (10 5 cells / well) on the middle layer.
  • FBS fetal bovine serum
  • RPMI 1640 medium 0.1% fetal bovine serum
  • the fluorescence of each well was read by omega (BMG Labtech, Ortenberg, Germany) and the start time for adhering to the medium layer was shown. After removal by gentle washing and aspiration at the indicated time points (60 and 120 minutes), the fluorescence of each well was imaged using Scion Image Software (Scion Corp., Frederick, Md.) To quantify the adherent cells Pixel.
  • Cell migration assays were performed in a Boyden chamber with a polycarbonate filter (pore size 8- ⁇ m) without polyvinylpyrrolidone (PVP). The filter was thoroughly washed with PBS and dried just before use. The same Boyden chamber was used for cell penetration analysis, but a polycarbonate filter pre-coated with Matrigel at a concentration of 1 g / ml was used. Cells were trypsinized and resuspended in RPMI 1640 containing 1% FBS and added to the upper chamber in the presence or absence of TGF-beta (5 ng / ml) for 24 or 36 hours. The same RPMI 1640 medium containing 5% FBS was added to the bottom chamber.
  • RPMI 1640 containing 5% FBS was added to the bottom chamber.
  • Cells were treated with paclitaxel (A.G. Scientific, San Diego, Calif.) At the indicated appropriate concentrations.
  • paclitaxel A.G. Scientific, San Diego, Calif.
  • cell viability was measured by MTT assay.
  • Cell death percentages were determined by double staining of annexin V and propidium iodide (PI), which measure the exposure of phosphatidylserine to the outside of the cell membrane, and cell membrane integrity during cell death was excluded. This was performed according to the manufacturer's protocol using the Annexin-V FITC Apoptosis Detection Kit (BioBud, Seongnam, Korea). The stained cells were analyzed by FACS cater-plus flow cytometer.
  • PI propidium iodide
  • Biotinylated synthetic nc886 was made with the MEGAscript® T7 transcription kit (Ambion, Waltham, MA) using biotin-16-UTP (Roche Applied Science, Indianapolis, IN).
  • a biotinylated oligoribonucleotide GE Dharmacon, Lafayette, CO having the tRNA sequence (5'-gaagcggggucucuuauuu-3 ') was synthesized and used in parallel.
  • Streptavidin magnetic beads were purchased from New England Biolabs (Ipswich, Mass.).
  • the S100 fraction Prior to binding to the nc886-beads, the S100 fraction was thawed, incubated at room temperature (RT) for 2 hours, and super-centrifuged (40,000 rpm at 40,000 rpm for 15 minutes on a Beckman TLA 100.3 rotor). The supernatant (“clear S100 fraction”) was preincubated with empty beads for 30 minutes at 4 ° C before incubation with experimental beads containing nc886 or tRNA fragments.
  • 293T cells were transfected with a plasmid expressing FLAG-Dicer ("pcDNA3.1-FLAG-Dicer (WT)" in [Table 3]). Approximately 10 7 cells were suspended in 1 ml Buffer D [20 mM HEPES-KOH (pH 7.4), 100 mM KCl, 0.2 mM EDTA, 5% glycerol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM DTT] -and-thaw and FLAG-Dicer were purified by pulling down with Anti-FLAG® M2 magnetic beads (Sigma-Aldrich, St. Louis, Mo.).
  • nc886 and vtRNA1-1 were synthesized with the MEGAscript® T7 transcription kit.
  • RNA gel-loading buffer (95% formamide, 18 mM EDTA, 0.025% sodium dodecyl sulfate).
  • the reaction products were separated on a 15% denaturing polyacrylamide gel and Northern hybridized using probes for each precursor (Table 4).
  • NCBI National Center for Biotechnology Information
  • GEO Gene Expression Omnibus
  • Gene expression data were generated using an Affymetrix microarray platform (U133 v2.0). All data were normalized using robust multi-array averaging. All patients underwent cytoreduction and platinum-based chemotherapy.
  • BRB-ArrayTools was used primarily to statistically analyze gene expression data, and all other statistical analyzes were performed in the R language environment (http://www.r-project.org). Cluster analysis was performed using Cluster and Treeview 55.
  • the development and validation of prediction models based on gene expression signatures and the estimation of prediction accuracy were performed as follows:
  • the expression pattern (training set) of the 118 genes obtained from the cell line was determined using a Bayesian compound covariate predictor (BCCP) Algorithms have been combined to form a classifier. This algorithm estimates the probability that a particular sample belongs to a subgroup.
  • the computational error rate of this training set was estimated by Leave-One-Out-Cross-Validation (LOOCV) classification and the classifier was applied directly to the gene expression data (test set) of ovarian cancer patients. Kaplan-Meier plots and log rank tests were used to predict patients' prognosis.
  • LOOCV Leave-One-Out-Cross-Validation
  • ROC receiver-operating characteristic
  • MS-HRM Bisulfite treatment and PCR reactions for MS-HRM were performed as described in the EpiTPYER assay. PCR primers were listed in Table 4 and amplicons were set in the range of nt -424 to +558 (-424 to -254, -294 to -141, -162 to -15, -40, +68, +45 to +207, +230 to +317, +293 to +443 and +469 to +558). Melting curves of MS-HRM were obtained on a LightCycler® 480 Instrument II and analyzed using GeneScanning software (Roche, Basel, Switzerland).
  • ChIP Chromatin Immunoprecipitation
  • ChIP was performed using the ChIP Assay Kit (EMD Millipore, Billerica, Mass.) According to the manufacturer's protocol. Briefly, formaldehyde (final concentration 1%) - fixed cells were harvested and lysed. The chromatin was sonicated to obtain 300-500 nucleotide DNA fragments and then immunoprecipitated at 4 ° C for 24 hours using anti-SMAD4 antibody (sc-73599 from Santa Cruz Biotechnology, Dallas, TX). The antibody-chromatin complex was pulled with salmon sperm DNA-protein G-agarose beads. After cross-linking was reversed, DNA (immunoprecipitation and injection) was purified using a Fragment DNA purification kit (Intron Biotechnology, Seoul, Korea) and PCR amplified using the primers shown in Table 4.
  • mice BALB / c thymic nude mice (Orient Bio, Seongnam, Korea) were used. All rats were 20-25 grams of females and were kept free of food and water under normal laboratory conditions.
  • TGF- ⁇ (1 ⁇ g per kg of body weight) from the fifth week after inoculation, and the remaining half were treated with vehicle PBS via peritoneal injection. Processing was done three times a week (Monday, Wednesday, Friday). At 8 weeks after inoculation, mice were sacrificed and dissected, and the number and location of individual tumor nodules were confirmed and recorded.
  • Luciferase assays were performed using the Dual-Luciferase® Reporter Assay System (Promega, Madison, Wis.). OSE80PC cells (96-well plate) were transfected with 5 ng of pRL-SV40 combined with 0.5 ng of the indicated sensor plasmid (Table 3) and miRNA mimic or control mimic RNA per well. Twenty-four hours after transfection, cells were harvested for luciferase analysis according to the manufacturer's instructions. The luciferase value was normalized and plotted as described in the prior art document 1.
  • the trypsin peptide was resuspended in 10 ⁇ l loading solution (5% methanol containing 0.1% formic acid) and loaded onto a Nano LC 1000 system (Thermo Fisher Scientific, Waltham, Calif.) Coupled to an LTQ Orbitrap Elite TM mass spectrometer (Thermo Fisher Scientific) MA) in a nanoflow LC-MS / MS assay.
  • the peptide was loaded onto a 2 cm x 100 mu m pre-column of Reprosil-Pur Basic C18 (1.9 mu m, Dr. Maisch GmbH, Germany).
  • the pre-column was switched in series with a 50 mm x 150 ⁇ m analytical column packed with Reprosil-Pur Basic C18 equilibrated in 0.1% formic acid / water.
  • the peptides were eluted using a 75 min discontinuous gradient of 4-26% acetonitrile / 0.1% formic acid at a flow rate of 600 nl / min.
  • the eluted peptides were directly electrosprayed into an LTQ Orbitrap Elite mass spectrometer operating in a data-dependent acquisition mode to obtain the splitting spectra of the top 50 strongest ions.
  • MS / MS spectra obtained were retrieved from the Proteome Discoverer 1.3 interface (Thermo Fisher Scientific) for the target-decoy human Refseq database using the Mascot algorithm (Mascot 2.3, Matrix Science).
  • the precursor mass tolerance was limited to within 20ppm, and the fragment mass tolerance was 0.5 daltons and allowed a maximum of 2 missing cuts. Oxidation, protein N-terminal acetylation and dynamic deformation of the breakdown were allowed.
  • the peptides identified in the mascot result file have been validated with a 5% false discover rate (FDR) and are subject to manual verification. The number of recovered peptides was used to calculate the amount of protein present to compare the relative amounts between different conditions of the sample.
  • FDR 5% false discover rate
  • nc886 was quantified after northern hybridization, and TGFBI was measured by qRT-PCR and expressed as a relative value to primary OSE. nc886 expression was higher in ovarian surface epithelial cells than in ovarian surface epithelial cells in OSE80PC, MPSC1, HeyA8 and OVCA5, while nc886 expression was silenced in A2780, SKOV3, OVCA433, OVCA432, IGROV-1 and BG-1.
  • nc886 expression was due to CpG DNA and methylation, which was treated with 5-Aza-2'deoxycytidine (AzadC), a post-translational modification that resulted in DNA demethylation restoring nc886 expression to SKOV3 and A2780 cells
  • Fig. 2 5-Aza-2'deoxycytidine
  • SKOV3 cells were treated with TGF- ⁇ .
  • nc886 and TGFBI were significantly increased, and expression of SMAD5 was increased but weak (Fig. 4).
  • the induction of nc886 by TGF- ⁇ was nullified by the ectopic expression of DNA methyl-transferase (DNMT1) (FIG. 4).
  • SKOV3 cells were transfected with DNMT1 expression plasmid (and vector control) 48 hours after treatment with 10 ng / ml of TGF-beta, and after 24 hours of harvesting the cells, the expression level of nc886 was measured by qRT-PCR Respectively.
  • nc886 by TGF- ⁇ is mediated through postmortem mechanisms of hypomethylation.
  • MS-HRM methylation-sensitive high resolution lysis
  • TGF- ⁇ treatment alters the dissolution profile similar to that of low-methylated DNA (nt -424 to -254, all nt numbers +1 at the 5 'end of the nc886 transcript).
  • individual CpG sites in the -165 to + 493 nt region were investigated by EpiTYPER analysis.
  • nc886 induction by TGF- ⁇ was not impaired.
  • TGF- ⁇ induced SMAD4 binding in the nc886 genome region Did not do it. It was also confirmed that the induction was not due to a change in the number of genome copies.
  • nc886 is an ncRNA induced by TGF- ⁇ through a posterior mechanism.
  • SKOV3 and A2780-derived ovarian cancer cell lines stably expressing nc886 were generated.
  • the expression level of ectopic nc886 was not higher than the endogenous expression level of primary OSE cells or OSE80PC cells and was similar to that of TGF- ⁇ -induction (FIG. 6), confirming that the phenotype of the stable cell clone was not an artificial result.
  • nc886 an ncRNA induced by TGF- ⁇ .
  • Ectopic expression of nc886 in SKOV3 and A2780 cells promoted adhesion to mesothelial cells and migration and invasion ([Figure 7] - [ Figure 9]) similar to TGF- ⁇ treatment.
  • Data from in vitro assays were supported by in vivo experiments.
  • SKOV3_vector or SKOV3_nc886 cells were inoculated intraperitoneally into BALB / c thymic nude mice and treated with TGF-? Or phosphate buffered saline as a vehicle control via intraperitoneal injection. As a result, it was confirmed that when nc886 was expressed and TGF- ⁇ was treated, SKOV3 cells were transferred to distant organs (FIG. 12).
  • the TGF-beta pathway may exhibit multiple effects, so it may be desirable to use an antisense oligonucleotide (anti-nc886 or nc886-kd) as well as a control anti-oligo ("anti- Or "control-kd”) to evaluate the role of nc886.
  • anti-nc886 or nc886-kd an antisense oligonucleotide
  • control-kd anti- Or "control-kd”
  • nc886 plays an important role in TGF- ⁇ induced ovarian cancer metastasis.
  • paclitaxel treatment enriched the annexin V-positive (apoptosis) cells.
  • apoptosis apoptosis
  • the fraction of apoptotic cells was similar between the four experimental sets (nc886 and / or TGF-?).
  • nc886 and / or TGF- ⁇ elicited significant differences in MTT levels or basal levels of apoptosis.
  • paclitaxel induces apoptosis of ovarian cancer cells, while nc886 / TGF-beta confers resistance to paclitaxel.
  • nc886 mimics gene expression expression mediated by TGF- [beta]
  • TGF- [beta] global gene expression by mRNA microarrays was measured.
  • fc multiple of the expression value
  • 380 genes were transformed ("SKOV3_nc886" versus "SKOV3_vector").
  • OSE80PC cells were transfected with 100 nM of nc886 target anti-oligo ("anti-nc886”) or non-target anti-control ("anti-control"). Cells were harvested 48 hours after transfection for nuclear / cytosolic fractionation. Two sets of experiments were added with nc886-kd in OSE80PC cells and nc886 expression in A2780 cells. Pair comparisons were performed with four data sets (SKOV3_TGF- ⁇ , SKOV3_nc886, A2780_nc886, and OSE80PC_nc886-kd) to determine whether there is a gene expression correlation.
  • nc886 can mimic TGF- ⁇ -mediated reprogramming of gene expression.
  • nc886 was an inhibitor of PKR, an activator of NF- ⁇ B, and TGF- ⁇ inhibited NF- ⁇ B, thus confirming the NF- ⁇ B pathway.
  • NF- ⁇ B and related pathways (IL1R_PATHWAY, NFKB_PATHWAY, NTHI_PATHWAY, CD40_PATHWAY) were evaluated as the most inhibited pathway in TGF- ⁇ treated SKOV3 cells.
  • nc886 induced by TGF- [beta] inhibits the activity of PKR and consequently NF-kB.
  • nc886 did not have a significant effect on NF- ⁇ B and related pathways.
  • PKR was actually activated in the form of phospho-PKR (the active form of PKR), resulting in phosphorylation of its substrate eIF2 ⁇ .
  • NF- ⁇ B was not activated, suggesting that PKR / NF- ⁇ B binding is irrelevant in OSE80PC cells.
  • the phospho-PKR band (where nc886 was reduced or silenced) was not stronger than the basal expression of OSE80PC cells (where nc886 expression was high).
  • nc886 miRNA Suppress the path.
  • nc886 is exclusively localized to the cytoplasm (Fig. 15), there will be little direct role of nuclear events such as TF activity and chromatin remodeling. Therefore, we investigated the effect of nc886 on gene expression.
  • MIRs miRNA target gene set
  • MSigDB miRNA target gene set
  • nc886 and TGF- ⁇ influence the overall MIR pattern.
  • the MIR Z score was calculated for each pair of experiments and the 221 values were sorted from the lowest value to the highest value and the data plotted ( Figure 20).
  • the Z-score distribution was shifted down with the MIR set at position 190 where x-intercept was. This indicates that 189 MIRs have been depleted but only 31 MIRs have been abundant. That is, the activity / level of most miRNAs increased at nc886kd.
  • TGF- ⁇ treatment and nc886-high
  • the pattern was reversed: 27 and 193 MIRs were depleted and enriched.
  • TFT TF target gene set
  • MSigDB TF target gene set
  • TFT sets 615 TFT sets, each of which has a target gene for TF.
  • TFTs 615 TFT sets, each of which has a target gene for TF.
  • Some TFTs were abundant and other TFTs were depleted.
  • the overall ranking distribution is relatively balanced.
  • the tendency of MIRs for positive values also appeared in ectopic expression of nc886 in SKOV3 and A2780 (data not shown); However, the size was lower in TGF- ⁇ treatment. It was assumed that the cell line was stable and that the direct effect of nc886 on MIR was diffused during the long - term culture. Therefore, further analysis focused on nc886-kd and TGF- ⁇ treatment. Overall, nc886 levels were positive for MIR, indicating that nc886 inhibits the miRNA pathway.
  • the MIR Z-scores of nc886-kd and TGF- ⁇ are shown in the scatter plot.
  • the TFT Z-score was plotted in the same manner.
  • MIR scatter plot most data points are confined to the second quadrant, showing that MIR generally decreases and increases by nc886-kd and TGF- ⁇ .
  • SKOV3_TGF- ⁇ SKOV3_nc886
  • A2780_nc886 OSE80PC_nc886-kd
  • the MIR correlation was stronger than the TFT correlation.
  • the scatter plot between "SKOV3_TGF- ⁇ ” and "SKOV3_nc886” and between SKOV3_nc886 and OSE80PC_nc886-kd showed positive and negative correlations, respectively.
  • the MIR profile was examined to identify miRNAs most affected by TGF-beta / nc886.
  • the MIR was classified according to the sum of the Z scores in the four experimental sets. Richly replicated miRNAs were selected from the top-level MIR-associated miRNAs (based on miRbase, http://www.mirbase.org/), and finally 5 miRNAs (miR-124-3p, -183-5p , -203a-3p, -200c-3p and -19b-3p) were selected and they were shown to inhibit ovarian cancer cell motility.
  • miRNA target genes were searched in the nc886 / TGF-beta pathway in ovarian cancer (Fig. 23). We considered 1552 array probes that were initially significantly altered (p ⁇ 0.05) in both nc886-kd and TGF- ⁇ treatments and selected 477 probes that decreased in both nc886-kd and TGF- ⁇ . The effect of miRNA on the target mRNA is usually mild, so no fc cutoff was applied. When 397 mRNA genes of miRNA target candidates, probes for probes overlapping ncRNA and tin-free genes are removed, 397 mRNA genes are generated.
  • the genes targeted by one or more miRNAs were selected to further probe the expected miRNA target. This is because several miRNAs that inhibit a single gene are apparently more inhibited than single miRNAs through cooperative or simple weighting. As a result, it was confirmed that over 40 miRNAs targeted 13 genes, some of which were known to be associated with ovarian cancer (Fig. 24). Among them, CNN3, PDCD6, PRKCA, and ZEB2 were selected as additional test subjects. qRT-PCR measurements confirmed that their expression was reduced by nc886-kd and increased by TGF-beta ( Figure 24), consistent with the sequence data.
  • miRNA-mimic (a mixture of miR-124-3p, -183-5p, -203a-3p, -200c-3p and -19b-3p) was tested in OSE80PC cells (nc886- miRNAs are low). The levels of all three genes except PRKCA were reduced by miRNA mimetics (Figure 29). Next, it was confirmed whether CNN3, PDCD6 and ZEB2 were direct targets by performing luciferase analysis with a sensor plasmid having a 3'-untranslated region (UTR) of these genes (Fig. 29). All three sensor plasmids were direct target genes in response to miRNA mimetics (Figure 29).
  • nc886 regulates key portions of hundreds of miRNAs, and that each miRNA has some effect on hundreds of targets, nc886 is thought to inhibit total miRNA activity.
  • nc886 By interacting with Dicer miRNA It inhibits maturation.
  • nc886 Unlike most regulated ncRNAs that recognize target DNA or RNA and regulate gene expression, nc886 appears to interact by interacting with proteins and modulating their activity, as seen in the nc886 / PKR case. Therefore, the target protein of nc886 was identified in ovarian cancer. To do this, we used in vitro biotinylated nc886 to search for proteins that interact with nc886 in the soluble cytoplasmic extract of OSE80PC cells (S100 fraction).
  • ncRNA1-1 is similar to nc886 in length (99 nt vs. 101 nt) and sequence (38 identical nts when 6 consecutive identical nucleotides are calculated). Nevertheless, these ncRNAs bind differently than Dicer. Wild-type and mutant Dicer ("pcDNA3.1-FLAG-Dicer-" series) or pcDNA3.1-FLAG vectors were transfected with 293T cells.
  • nc886 was present in FLAG-Dicer IP complex at a significantly higher level than vtRNA1-1 (WT in [Fig. 26]).
  • WT in [Fig. 26]
  • vtRNA1-1 shares most of its other characteristics, such as intracellular abundance, cytoplasmic location, and transcription by Pol III, thus requiring optimal control.
  • vtRNA1-1 was not significant for expression of the tumor or miRNA target gene (data not shown), which is in contrast to nc886. It is assumed that Dicer binds to nc886 and vtRNA1-1, respectively, because it is proficient and incomplete.
  • the nc886-Dicer interaction was demonstrated by testing truncation mutations (Figure 25).
  • the mechanism of Dicer interaction for pre-miRNA has been intensively studied in many literature.
  • the PAZ (PIWI-AGO-ZWILLE) domain recognizes the end of the pre-miRNA and plays an important role in determining its position in the RNase III catalytic domain ("RIIIDa” and "RIIIDb” in [FIG. 25]).
  • the ATPase / helicase domain (“Helicase” in [ Figure 25]) interacts with the pre-miRNA loop and facilitates the processing of some pre-miRNAs.
  • DUF283 a domain of unknown function
  • dsRBD double-stranded RNA binding domain
  • nc886 interaction in FLAG-IP data decreases in the absence of helicase domains (WT and ⁇ DUF comparisons) and further decreases ( ⁇ DUF and ⁇ PAZ comparisons) when the PAZ domain is deleted.
  • WT and ⁇ DUF comparisons helicase domains
  • ⁇ DUF and ⁇ PAZ comparisons when the PAZ domain is deleted.
  • the nc886 signal was above the background level (FLAG) but could not be distinguished from the vtRNA1-1 signal.
  • nc886-Dicer interaction induced damaged miRNA processing based on the fact that mature miRNA levels are increased / decreased when nc886 is low / high (Fig. 22).
  • competitor unlabeled nc886 or vtRNA1-1
  • miR-124-1 and -200c precursors were efficiently processed into mature miRNAs by FLAG-purified Dicer.
  • nc886 efficiently inhibits miRNA maturation when added to the processing assay as compared to vtRNA1-1 ( Figure 27).
  • This data is in good agreement with the binding data ( Figure 26), so it is likely that nc886 binds to Dicer and as a result, it can titrate Dicer from the true miRNA precursor.
  • nc886 not only binds but is degraded by Dicer ( Figure 27).
  • Degradation of nc886 by Dicer also occurred in ovarian cancer cells as shown by our experiments, indicating that ectopic expression of Dicer and kd result in reduced expression levels and increased expression levels of nc886, respectively (Fig. 28).
  • nc886 The degradation of nc886 was distinguished from real miRNA processing. Unlike a single discrete band of mature miRNA size (22 nts) of true pre-miRNA, Dicer degrades nc886 into multiple bands in a wide range of 25-80 nts (Figure 27). nc886 barely produced mature miRNA in this assay consistent with mature miR-886 detectable in whole cell RNA. The degree of nc886 degradation is quantitatively less than the amount of pre-miR-200c treatment (Fig. 27). Canonical pre-miRNAs have a nearly complete duplex stem with a 2 nt 3'-overhang end structure. This region is recognized by the PAZ domain which correctly positions the cleavage site in the catalytic core.
  • nc886 lacks such structural features to account for less efficient wobble cutting. Although nc886 is a weaker substrate than the true pre-miRNA, nc886 is expected to be able to compete with Dicer because it is highly abundant in cells (10 5 copies / cell). nc886 interacts physically with Dicer, acts as a pseudo-substrate, and inhibits the miRNA pathway by titrating away from miRNA precursors.
  • nc886 The phenotype is due to Dicer inhibition.
  • Dicer inhibition was then assessed.
  • SKOV3 vector cells were treated with TGF-beta for 96 hours, transfected with pcDNA3.1-FLAG-Dicer (WT) for 24 hours and analyzed.
  • Dicer kd decreased mature miRNA levels and enhanced cell adhesion, similar to that performed by TGF- ⁇ and nc886 (FIG. 30).
  • ectopic expression of Dicer attenuated cell migration stimulated by TGF-beta or nc886 ( Figure 31).
  • the inhibitory effect of Dicer on cell migration was not nonspecific toxic effect because PARP level was the same and ectopic expression of Dicer did not inhibit SKOV3_vector. This clearly demonstrated that Dicer inhibition is an important event in the role of TGF- ⁇ / nc886 in promoting cell metastatic potential.
  • nc886 In ovarian cancer patients, chemotherapy resistance (chemical resistance), poor prognosis Be relevant .
  • nc886 was altered in our model of inhibiting the miRNA pathway to increase miRNA target genes ("nc886_kd” decreased and "nc886_exp” or "TGF- ⁇ ” increased).
  • nc886_kd decreased and "nc886_exp” or "TGF- ⁇ ” increased.
  • nc886 did direct the clinical outcome of patients with ovarian cancer after treatment, and as shown in Table 6, in the univariate and multivariate Cox regression analysis, the nc886 subtype was significantly associated with overall survival,
  • clinical data are in good agreement with our cell culture data (Fig. 11 and [Fig. 35]), indicating that novel TGF-beta target genes and miRNAs Regulator Nc886 of the tumor can be a major predisposing factor for ovarian cancer shows comprehensively that it can be the target of future chemotherapy.
  • the present invention can prevent or treat ovarian cancer by inhibiting the expression of nc886 gene, and can diagnose ovarian cancer by measuring the expression level of nc886 gene.
  • the present invention is effective as an information providing method for ovarian cancer prognosis prediction by analyzing the expression level of nc886 to predict the ovarian cancer metastasis or resistance to an anticancer agent.
  • SEQ ID No. 1 of the present invention represents the sequence of nc886.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une composition qui permet de diagnostiquer, de prévenir ou de traiter le cancer de l'ovaire sur la base du niveau d'expression du gène nc886. La présente invention concerne également un procédé de fourniture d'informations pour la prédiction du pronostic pour des patientes souffrant d'un cancer de l'ovaire par la confirmation du sous-type de cancer de l'ovaire et du niveau d'expression du gène nc886 chez les patientes souffrant d'un cancer de l'ovaire. Le procédé de fourniture d'informations selon l'invention comprend un procédé de fourniture d'informations visant à identifier le niveau d'expression du gène nc886 avec des motifs d'expression d'autres gènes associés au gène nc886. La présente invention concerne également l'utilisation d'une composition pour la prédiction d'un pronostic pour le cancer de l'ovaire, la composition comprenant un agent pour la mesure du niveau d'expression du gène nc886. Par conséquent, la présente invention peut présenter un effet de prévention ou de traitement du cancer de l'ovaire par inhibition de l'expression du gène nc886 et permet de diagnostiquer le cancer de l'ovaire ou de prédire le pronostic pour des patientes souffrant d'un cancer de l'ovaire par mesure du niveau d'expression du gène nc886. De plus, la présente invention analyse le niveau d'expression du gène nc886 pour prédire la métastase ou la résistance du cancer de l'ovaire à un agent anticancéreux et se révèle donc efficace en tant que procédé de fourniture d'informations pour la prédiction d'un pronostic pour le cancer de l'ovaire.
PCT/KR2018/013126 2017-10-31 2018-10-31 Procédé de fourniture d'informations pour la prédiction d'un pronostic pour le cancer de l'ovaire utilisant le gène nc886 WO2019088709A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20170144330 2017-10-31
KR10-2017-0144330 2017-10-31

Publications (2)

Publication Number Publication Date
WO2019088709A2 true WO2019088709A2 (fr) 2019-05-09
WO2019088709A3 WO2019088709A3 (fr) 2019-06-27

Family

ID=66333411

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/013126 WO2019088709A2 (fr) 2017-10-31 2018-10-31 Procédé de fourniture d'informations pour la prédiction d'un pronostic pour le cancer de l'ovaire utilisant le gène nc886

Country Status (2)

Country Link
KR (1) KR102082462B1 (fr)
WO (1) WO2019088709A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20240041396A (ko) * 2022-09-22 2024-04-01 차의과학대학교 산학협력단 파클리탁셀에 대하여 내성을 갖는 난소암의 진단을 위한 분석방법

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2354246A1 (fr) * 2010-02-05 2011-08-10 febit holding GmbH ARNm dans le diagnostic du cancer ovarien
WO2012054824A2 (fr) * 2010-10-22 2012-04-26 Vermillion, Inc. Biomarqueurs pronostiques chez des patientes atteintes d'un cancer ovarien

Also Published As

Publication number Publication date
KR20190049609A (ko) 2019-05-09
KR102082462B1 (ko) 2020-02-27
WO2019088709A3 (fr) 2019-06-27

Similar Documents

Publication Publication Date Title
Li et al. Long non-coding RNAs AC026904. 1 and UCA1: a “one-two punch” for TGF-β-induced SNAI2 activation and epithelial-mesenchymal transition in breast cancer
Mondal et al. Sense-antisense lncRNA pair encoded by locus 6p22. 3 determines neuroblastoma susceptibility via the USP36-CHD7-SOX9 regulatory axis
Li et al. Long non-coding RNA linc00460 promotes epithelial-mesenchymal transition and cell migration in lung cancer cells
Rong et al. Ribosome 18S m6A methyltransferase METTL5 promotes translation initiation and breast cancer cell growth
Duan et al. TET1 inhibits EMT of ovarian cancer cells through activating Wnt/β-catenin signaling inhibitors DKK1 and SFRP2
Choi et al. miR-93/miR-106b/miR-375-CIC-CRABP1: a novel regulatory axis in prostate cancer progression
Otsubo et al. MicroRNA-126 inhibits SOX2 expression and contributes to gastric carcinogenesis
Tsitsipatis et al. AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production
Perdigao-Henriques et al. miR-200 promotes the mesenchymal to epithelial transition by suppressing multiple members of the Zeb2 and Snail1 transcriptional repressor complexes
Zhou et al. The aberrantly expressed miR-193b-3p contributes to preeclampsia through regulating transforming growth factor-β signaling
Li et al. MicroRNA-25 functions as a potential tumor suppressor in colon cancer by targeting Smad7
Zhang et al. KLF5 activates microRNA 200 transcription to maintain epithelial characteristics and prevent induced epithelial-mesenchymal transition in epithelial cells
Ugalde et al. Aging and chronic DNA damage response activate a regulatory pathway involving miR‐29 and p53
Chung et al. Dysregulation of microRNA‐204 mediates migration and invasion of endometrial cancer by regulating FOXC1
Chen et al. MicroRNA-24 increases hepatocellular carcinoma cell metastasis and invasion by targeting p53: miR-24 targeted p53
Inaguma et al. GLI1 interferes with the DNA mismatch repair system in pancreatic cancer through BHLHE41-mediated suppression of MLH1
Tu et al. Circ-0001313/miRNA-510-5p/AKT2 axis promotes the development and progression of colon cancer
Yamamoto et al. Uncovering direct targets of MiR-19a involved in lung cancer progression
Su et al. miR‑885‑5p upregulation promotes colorectal cancer cell proliferation and migration by targeting suppressor of cytokine signaling
Petrie et al. Effects on prostate cancer cells of targeting RNA polymerase III
Fu et al. A loss of insulin-like growth factor-2 imprinting is modulated by CCCTC-binding factor down-regulation at senescence in human epithelial cells
Ai et al. lncRNA TUG1 promotes endometrial fibrosis and inflammation by sponging miR-590-5p to regulate Fasl in intrauterine adhesions
Gong et al. lncRNA FEZF1‑AS1 contributes to cell proliferation, migration and invasion by sponging miR‑4443 in hepatocellular carcinoma
Abukiwan et al. Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-β-driven progression of pancreatic cancer Corrigendum in/10.3892/ijo. 2023.5543
WO2021241980A1 (fr) Composition pour la prédiction de pronostic de cancer et kit la comprenant

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18872614

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18872614

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 18872614

Country of ref document: EP

Kind code of ref document: A2