WO2018133733A1 - 一种靶向rna解旋酶dhx33的小分子rna及其应用 - Google Patents

一种靶向rna解旋酶dhx33的小分子rna及其应用 Download PDF

Info

Publication number
WO2018133733A1
WO2018133733A1 PCT/CN2018/072441 CN2018072441W WO2018133733A1 WO 2018133733 A1 WO2018133733 A1 WO 2018133733A1 CN 2018072441 W CN2018072441 W CN 2018072441W WO 2018133733 A1 WO2018133733 A1 WO 2018133733A1
Authority
WO
WIPO (PCT)
Prior art keywords
dhx33
cancer
plasmid
nucleic acid
lentivirus
Prior art date
Application number
PCT/CN2018/072441
Other languages
English (en)
French (fr)
Inventor
张严冬
Original Assignee
深圳开悦生命科技有限公司
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
Priority claimed from CN201710049673.XA external-priority patent/CN108342415B/zh
Priority claimed from CN201710049813.3A external-priority patent/CN108339126B/zh
Priority claimed from CN201710049669.3A external-priority patent/CN108338986B/zh
Priority claimed from CN201710049975.7A external-priority patent/CN108342365B/zh
Application filed by 深圳开悦生命科技有限公司 filed Critical 深圳开悦生命科技有限公司
Publication of WO2018133733A1 publication Critical patent/WO2018133733A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

Definitions

  • the present application relates to the field of biotechnology, and in particular to a small RNA targeting a RNA helicase DHX33, a nucleic acid comprising a nucleic acid sequence encoding the small RNA or its complementary sequence, containing the small RNA or the nucleic acid sequence Lentiviral plasmids and lentiviruses, and their use in the treatment of cancer, especially lung adenocarcinoma.
  • c-Myc is one of the oncogenes highly expressed in many cancers, which can cause gene amplification or translocation in various cancers. Meanwhile, Myc inhibitory protein Mga gene deletion mutation can also cause overexpression of Myc. . c-Myc binds to Max mainly in cells, forming a transcription factor of c-Myc/Max dimer complex, which functions to regulate downstream genes and then stimulate cell proliferation and metabolism. The dysfunction of this function occurs in almost all human cancers. . In response to cancer cells' oncogene addiction, the administration of drugs targeting different oncoproteins can specifically inhibit the development of cancer, prolong the life of patients, and achieve precise personalized treatment.
  • EGFR-TKI EGFR tyrosine kinase receptor inhibitor
  • the purpose of the present application is to provide a novel drug capable of treating cancer caused by amplification or activation of the oncogene c-Myc.
  • the present application relates to a small molecule RNA targeting DHX33 or a complement thereof, characterized in that the small molecule RNA comprises the sequence:
  • DHX33 is a member of the RNA helicase family and is involved in protein translation and ribosomal RNA production, and is actively involved in the inhibition of apoptosis.
  • DHX33 is a gene downstream of c-Myc and plays a crucial role in c-Myc-induced cancer development.
  • the small RNA can knock down the protein content of DHX33 to a basic normal level, reduce the carcinogenicity of c-Myc and maintain the cancerous characteristics, thereby achieving the purpose of treating cancer.
  • the present application relates to a nucleic acid comprising a nucleic acid sequence encoding the small molecule RNA or a complement thereof, characterized in that the nucleic acid sequence is the sequence:
  • sh-DHX33-2 5'-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCC AACCAGCTTCCCAA-3'.
  • the application relates to a lentiviral plasmid comprising the small molecule RNA or the nucleic acid sequence.
  • the application relates to a lentivirus comprising the small molecule RNA or the nucleic acid sequence.
  • Fig. 1 is a graph showing the results of detection of protein content in Example 1 of the present invention.
  • Fig. 2 is a view showing the results of detection of apoptosis of cells observed under a microscope in Example 1 of the present invention.
  • Fig. 3 is a graph showing apoptosis analysis by flow cytometry in Example 1 of the present invention.
  • Fig. 4 is a graph showing the results of detecting the protein content of BT549 in Example 2 of the present invention.
  • Fig. 5 is a graph showing the results of detection of the protein content of H1299 in Example 2 of the present invention.
  • Fig. 6 is a graph showing the results of detecting the levels of the apoptosis gene Bcl-2 and BAD messenger RNA in Example 2 of the present invention.
  • Figure 7 is a diagram showing the results of electrophoresis of PCR products of the chromosomal immunoprecipitation assay in which DHX33 binds to the Bcl-2 and BAD gene promoters in Example 2 of the present invention.
  • Figure 8 is a graph showing the results of real-time fluorescent PCR detection of the amount of DHX33 mRNA in Example 3 of the present invention.
  • Fig. 9 is a graph showing the results of detection of the amount of DHX33 protein in Example 3 of the present invention.
  • Figure 10 is a schematic diagram showing the respective bases in the conserved region E-box in which the c-Myc transcription factor binds to the promoter DNA in Example 3 of the present invention.
  • Figure 11 is a schematic diagram showing the E-Box sites and sequences contained in the proximal promoter of the DHX33 gene in Example 3 of the present invention.
  • Figure 12 is a graph showing the results of analysis of the change in the electrophoretic mobility rate of the direct binding of the c-Myc protein to the DHX33 gene promoter in Example 4 of the present invention.
  • Figure 13 is a graph showing the expression of DHX33 and the expression of c-Myc in the examples of normal lung tissue and lung cancer tissue in Example 4 of the present invention.
  • Figure 14 is a graph showing the analysis of the amount of each protein in Example 4 of the present invention.
  • Figure 15 is a graph showing the results of cell migration experiments in Example 4 of the present invention.
  • Figure 16 is a graph showing the results of BrdU cell proliferation assay in Example 4 of the present invention.
  • Figure 17 is a comparison diagram of a soft agar suspension independent growth experiment of cells in Example 4 of the present invention.
  • Figure 18 is a comparative diagram showing the inhibition of cell migration induced by deletion of DHX33 in lung adenocarcinoma cells in Example 4 of the present invention.
  • Figure 19 is a comparative diagram showing the ability of a control group and an experimental group to form tumors in mice in Example 5 of the present invention.
  • Fig. 20 is a graph showing the results of quantitative analysis of tumor formation in nude mice in the control group and the experimental group in Example 5 of the present invention.
  • Figure 21 is a graph showing the results of analyzing the inhibition of DHX33 protein expression by various small RNA molecules targeting DHX33 in Example 6 of the present invention.
  • treating includes limiting, slowing, halting, or reversing the progression or severity of an existing condition or condition.
  • the term "patient” refers to a mammal, such as a mouse, guinea pig, rat, dog or human, preferably a human.
  • RNA helicase DHX33 which is a small hairpin RNA comprising the following sequences:
  • sequence of the small RNA molecule is the following sequence:
  • DHX33 is a target site for the treatment of c-Myc-induced cancer
  • DHX33 regulation is one of the downstream pathways of the oncogene c-Myc
  • the downstream signaling pathway of the gene c-Myc prevents the onset and progression of cancer.
  • the small RNA molecule has the effect of knocking down the protein content of DHX33 to a substantially normal level, which can reduce the carcinogenicity of c-Myc and maintain the cancerous characteristics, thereby achieving the purpose of treating cancer.
  • the present application also provides the use of the small molecule RNA or its complement sequence for the preparation of a medicament for the treatment of cancer, wherein the cancer is a cancer caused by amplification or activation of the oncogene c-Myc.
  • the cancer is lung adenocarcinoma.
  • the application also provides a nucleic acid comprising a nucleic acid sequence encoding a small molecule RNA of the invention or a complement thereof, the nucleic acid sequence being the sequence:
  • sh-DHX33-2 5'-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCC AACCAGCTTCCCAA-3'.
  • sequence of the nucleic acid is the sequence:
  • sh-DHX33-2 5'-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCC AACCAGCTTCCCAA-3'.
  • the nucleic acid of the present invention has the effect of knocking down the protein content of DHX33 to a substantially normal level, and can reduce the carcinogenicity of c-Myc and maintain the cancerous characteristics, thereby achieving the purpose of treating cancer, especially lung adenocarcinoma.
  • the present application also provides the use of a nucleic acid of the present invention in the manufacture of a medicament for the treatment of cancer, wherein the cancer is a cancer caused by amplification or activation of the oncogene c-Myc.
  • the cancer is lung adenocarcinoma.
  • the nucleic acid sequence of the present invention can be introduced into a cell by an existing lentivirus to treat a cancer caused by amplification or activation of the oncogene c-Myc, particularly lung adenocarcinoma.
  • the application also provides a lentiviral plasmid, wherein the lentiviral plasmid comprises the small RNA.
  • the application also provides a lentiviral plasmid, wherein the lentiviral plasmid comprises a nucleic acid sequence of the invention:
  • sh-DHX33-2 5'-TTGGGAAGCTGGTTGGCTATACTCGAGTATAG CCAACCAGCTTCCCAA-3' (SEQ ID NO: 2).
  • the lentiviral plasmid of the invention comprises the following nucleic acid sequence:
  • sh-DHX33-2-pre-oligonucleotide 5'-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG 3',
  • sh-DHX33-2-post oligonucleotide 5'-AATTCAAAAATTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3'.
  • the lentiviral plasmid of the present invention contains a gene encoding a small RNA sequence targeting DHX33 or a complement thereof, which has the effect of knocking down the expression level of DHX33 protein.
  • the following lentiviral plasmids were used as experimental controls:
  • sh-DHX33-1 (5'-3'): GCTATCGCAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGC (SEQ ID NO: 1),
  • a lentiviral vector pLKO.1-vector was used to construct a lentiviral plasmid which was cloned into the pLKO.1 plasmid by restriction enzyme cleavage site AgeI/EcoRI, and it should be noted that pLKO is used in the present application.
  • the 1 plasmid is merely an example, and the present invention is not limited thereto.
  • sh-DHX33-1-pre-oligonucleotide 5'-CCGGGCTATCGCAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGCTTTTTG 3' (SEQ ID NO: 3);
  • sh-DHX33-1-post oligonucleotide 5'-AATTCAAAAAGCTATCG CAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGC 3' (SEQ ID NO: 4);
  • sh-DHX33-2-pre-oligonucleotide 5'-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG-3' (SEQ ID NO: 5);
  • sh-DHX33-2-post oligonucleotide 5'-AATTCAAAAA TTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3' (SEQ ID NO: 6);
  • the above sequence was cloned into the restriction enzyme site AgeI/EcoR of the pLKO.1 plasmid to obtain plasmid pLKO.1-shRNA.
  • the present application provides the use of the lentiviral plasmid of the present invention for the preparation of a medicament for treating cancer, wherein the cancer is a cancer caused by amplification or activation of the oncogene c-Myc.
  • the cancer is lung adenocarcinoma.
  • the application also provides a lentivirus comprising the lentiviral plasmid of the invention.
  • the lentivirus of the present invention has the effect of knocking down the expression level of DHX33 protein, and is a gene-targeted drug.
  • the lentivirus of the invention may comprise the small molecule RNA.
  • the lentivirus of the invention comprises a nucleic acid sequence encoding the small RNA or its complement:
  • sh-DHX33-2 5'-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCCAACCAGCTTCCCAA-3'.
  • the lentivirus of the invention comprises the following nucleic acid sequence:
  • sh-DHX33-2-pre-oligonucleotide 5'-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG 3',
  • sh-DHX33-2-post oligonucleotide 5'-AATTCAAAAATTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3'.
  • the lentiviral plasmid in the lentivirus is a pLKO.1 plasmid, and the nucleic acid sequence encoding the small RNA or its complement is cloned into pLKO.1 by restriction enzyme cleavage site AgeI/EcoRI In the plasmid.
  • the present application also provides the use of the lentivirus of the present invention for the preparation of a medicament for the treatment of cancer, wherein the cancer is a cancer caused by amplification or activation of the oncogene c-Myc.
  • the cancer is lung adenocarcinoma.
  • the present application also provides a method of treating cancer by knocking down the protein content of DHX33, which is a cancer caused by amplification or activation of the oncogene c-Myc.
  • the cancer is treated by administering a small molecule RNA of the invention or its complement to a patient.
  • the cancer is treated by administering a nucleic acid of the invention to a patient.
  • the cancer is treated by administering a lentiviral plasmid of the invention to a patient.
  • the cancer is treated by administering a lentivirus of the invention to a patient.
  • the application also provides a method of preparing a lentivirus of the invention, the method comprising the steps of:
  • 293T cells are cultured and mixed with a plasmid mixture when the 293T cells are grown to about 90% confluency, wherein the plasmid mixture comprises a lentiviral plasmid;
  • the medium was replaced with an antibiotic; after another 24 to 48 hours, the cell culture medium was collected, centrifuged at a low speed, and the supernatant was collected.
  • the plasmid mixture may comprise the lentiviral plasmid, the packaging helper plasmids pCMV-VSV-G and pCMV-dR8.2dvpr, and the mixing ratio may vary within a wide range, preferably about 9:8:1.
  • the rotational speed may vary over a wide range, preferably from about 1000 to 2000 rpm, and the time for low speed centrifugation is from 30 seconds to 10 minutes, preferably about two minutes.
  • Antibiotics are any antibiotics commonly used in the art, such as penicillin and/or streptomycin.
  • the lentivirus of the present invention can be prepared by the following method:
  • the plasmid mixture was transfected into 293T cells using Lipofectamine 2000 (Life Technologies) liposome introduction technique. Specifically, transfected with a 10 cm diameter cell culture dish, and when the 293T cells were grown to about 90% confluency, the plasmid pLKO.1-shRNA (ie, the lentiviral plasmid described above), the packaging helper plasmid pCMV-VSV-G, and pCMV-dR8.2dvpr was mixed therein, and the ratio of each plasmid was about 9:8:1, so that the total amount of DNA reached 12 micrograms per dish;
  • Lipofectamine 2000 Life Technologies
  • the application also provides a cell comprising the lentiviral plasmid of the invention or the lentivirus of the invention.
  • Example 1 Rapid degeneration of DHX33 in cancer cells leads to apoptosis
  • the small RNA sequence is a small RNA sequence targeting DHX33;
  • sh-DHX33-1 (5'-3'): GCTATCGCAAAGTGATCATTT (as a positive control group);
  • sh-DHX33-2 (5'-3'): TTGGGAAGCTGGTTGGCTATA.
  • Control group The small RNA (shScrambled) sequence as a negative control is:
  • the nucleic acid sequence encoding the above sequence was cloned into the restriction enzyme site AgeI/EcoRI of the pLKO.1 vector, respectively.
  • the oligonucleic acid was dissolved in water to obtain a solution having a concentration of 20 ⁇ mol, and the paired pre-oligonucleic acid and the post-oligonucleic acid were added to a sterile PCR tube in the following formula: 5 ⁇ l of each pre-oligonucleic acid , 5 ⁇ l of 10XNEB Buffer 2 (purchased) and 35 ⁇ l of water.
  • the positive clones were screened by agarose gel electrophoresis.
  • the positive clones had a 5KB and a 2KB band, and the sequencing analysis confirmed whether the nucleic acid sequence was correct.
  • lentivirus The plasmid mixture was transfected into 293T cells using Lipofectamine 2000 (Life Technologies) liposome introduction technique. The specific method is to use a 10 cm cell culture dish for transfection. When the 293T cells grow to 90% confluency, the plasmid pLKO.1-shRNA, the packaging helper plasmids pCMV-VSV-G and pCMV-dR8.2dvpr are mixed. Among them, the ratio of each plasmid was 9:8:1, and the total amount of DNA was 12 ⁇ g per dish. After 16-18 hours, replace it with medium containing antibiotics penicillin and streptomycin.
  • the cell culture medium After 24 or 48 hours, collect the cell culture medium with a sterile pipette, centrifuge at low speed, rotate at 2000 rpm, and the time is two. In minutes, the virus was dispensed into a 5 ml sterile centrifuge tube and stored in a refrigerator at minus 80 degrees.
  • Breast cancer cell lines BT549 and HCC1806 were obtained from ATCC in the United States. These two cells were each infected with a lentivirus containing a nucleic acid sequence encoding a small hairpin RNA, and after 72 hours, the cells were collected and total protein was extracted.
  • the specific method for extracting the protein is to suspend the cells in a cell lysate (20 mmol of Tris-HCl, 150 mmol of NaCl, 1 mmol of EDTA, 1% Triton-X-100, 1% SDS, and supplement) Inhibitors of proteases and phosphatases).
  • a stable and stable expression cell line of DHX33 was constructed. The expression level of DHX33 protein in each sample was analyzed by immunoblotting, and the total protein loading was analyzed with an anti-GAPDH antibody.
  • the results are shown in Figure 1.
  • the DHX33 stable expression system shSCR-BT549 and shSCR-HCC1806 in the control group can detect DHX33 protein; the experimental group DHX33 stable deletion system 1-sh-DHX33-BT549, 1-sh-DHX33-HCC1806, The DHX33 protein was almost undetectable in 2-sh-DHX33-BT549 and 2-sh-DHX33-HCC1806.
  • DHX33 was deleted from cancer cells, and the cell death was observed under a microscope.
  • the control cells shCCR-BT549 and shSCR-HCC1806 without DHX33 were vigorously grown and highly confluent; and the two groups of cells lacking DHX33 were 1-sh-DHX33-BT549 and 1-sh-DHX33-HCC1806.
  • 2-sh-DHX33-BT549 and 2-sh-DHX33-HCC1806 showed very obvious death. It is indicated that the loss of DHX33 in cancer cells can quickly lead to cell death.
  • the cells were further stained with Annexin V.
  • the specific method is to suspend the cells in phosphate buffer (137 mmol of NaCl, 2.7 mmol of KCl, 10 mmol of Na 2 HPO 4 , 1.8 mmol of KH 2 PO 4 ) to a concentration of 1*10 6 per ml. Cells. Then, Annexin V-FITC was added to the suspension of the cells, and after 15 minutes at room temperature, the cells were filtered through a 70 ⁇ m filter and analyzed by flow cytometry. The results are shown in Figure 3. The cells showed significant apoptosis after deletion of DHX33, and the apoptosis rate of the control group was small.
  • Example 2 DHX33 regulates important genes Bcl-2 and BAD that control apoptosis
  • Apoptosis is a process of programmed death in which cells undergo changes in the external or intrinsic environment. Mitochondrial proteins play an important role in the process of apoptosis caused by internal factors. Bak/Bax on the mitochondrial inner membrane forms a polymer channel that releases cytochrome c from the mitochondria into the cytoplasm, which in turn triggers the formation of apoptotic bodies and activation of caspase, which in turn causes irreversible series of protease cleavage reactions and cells. Apoptosis. Two proteins regulate the polymerization or activation of Bak/Bax, Bcl-2 inhibits Bak/Bax activation, while BAD inhibits Bcl-2 activity. Cancer cells generally have higher levels of Bcl-2 protein or lower levels of BAD, thereby inhibiting apoptosis.
  • DHX33 has a regulatory effect on the genes controlling BAD and BCL-2.
  • PCR primers used are as follows:
  • DHX33 protein directly regulates the transcription of BAD and Bcl-2 genes.
  • chromosome immunoprecipitation we used chromosome immunoprecipitation to analyze whether DHX33 binds to the promoters of BAD and Bcl-2.
  • the cells were then lysed, specifically with 2 ml of lysate containing 1% SDS, 50 mmol of Tris-HCl, 2 mmol of EDTA, 150 mmol of NaCl, and protease and phosphatase inhibitors. After the cells were lysed on ice for 10 minutes, they were fully lysed with a 40% output sonicator, and the long-stranded DNA was cut to a size of 500 bp to 1000 bp.
  • the mixture was incubated at 4 ° C overnight, and the antibody was bound to the antigen, and then 25 ⁇ l of Protein A-Sepharose was added, and the antibody-binding protein A-Sepharose was mixed at 4 ° C for one and a half hours, and then the supernatant was removed by centrifugation.
  • the pellet was washed 5 times with RIPA buffer containing 0.5 moles of NaCl.
  • the immunoprecipitate was then eluted with an eluent (0.2 moles of NaOH, 1% SDS), 100 microliters each time, for a total of 2 washes. 6 mol of NaCl was added to the eluted solution to a concentration of 0.6 mol. It was then treated overnight at 65 degrees Celsius to crosslink the DNA and protein. After extracting the DNA fragment therefrom using the PCR product extraction kit, the efficiency of binding of DHX33 to the BAD and Bcl-2 promoters was analyzed by PCR.
  • DHX33 directly regulates the transcription of these two genes as a transcription factor.
  • Example 3 c-Myc can positively regulate mRNA and protein expression of DHX33
  • FIG. 10 The c-Myc oncogene recognizes a specific base sequence as a transcription factor. These specific base sequences are named E-box. As shown in the figure, the conserved region of the c-Myc transcription factor binding to the promoter DNA E-box A conserved type of each base (Y-axis).
  • FIG. 11 A number of E-boxes near the proximal promoter of DHX33 were found by analysis. As shown, the DHX33 gene proximal promoter contains E-Box sites and sequences. These analyses indicated that c-Myc binds directly to the promoter of DHX33 and regulates gene transcription of DHX33.
  • Figure 12 EMSA assay to analyze whether c-Myc protein binds directly to the promoter of DHX33.
  • the 2 kb promoter region of the DHX33 promoter pGL3control plasmid (purchased) was cloned, and then the three E-boxes of DHX33 were mutated with the apex mutagenesis kit (purchased) to obtain the wild-type and mutant DHX33 promoters.
  • 5'-CTAGCTAGCTAGTTTGGACAGAGAAGGGGAAAAC-3' (SEQ ID NO: 15)
  • 5'-CCGCTCGAGCGGCCCTCTCAGGTGCAGACAAC-3' (SEQ ID NO: 16) were used as primers, and PCR was prepared.
  • the probe is labeled and then purified using a PCR product purification kit.
  • the probe to be labeled is heated at 95 ° C for 2 minutes, and immediately ice bath is made into a single chain;
  • c-Myc may be an upstream regulatory gene of DHX33, and co-stained c-Myc and DHX33 in lung cancer tissues.
  • Figure 13 shows The expression of DHX33 is positively correlated with the expression of c-Myc in many lung cancer tissues.
  • the first normal tissue in the figure is normal lung tissue.
  • the second to fourth lines of lung cancer in the figure are Three lung cancer tissue examples are shown.
  • DHX33 is essential for the carcinogenicity and maintenance of cancerous characteristics of c-Myc
  • the protein content of DHX33 was knocked down to a basic normal level in the cell system overexpressing c-Myc, observed after DHX33 deletion, c- Whether the divisional growth of Myc overexpressing cells is inhibited.
  • the specific procedure was as follows: in NIH3T3 cells (ATCC, ATCC-CRL-1658), the cells were treated with c-Myc overexpression lentivirus, and then shSCR control (shScrambled) and shDHX33 were used in the case of c-Myc overexpression, respectively.
  • FIG. 14 The lentiviral treatment of (sh-DHX33-2) brought DHX33 to near normal levels.
  • Figure 14 is an analysis chart of the amount of each protein.
  • Figure 15 is an experimental result of analyzing the mobility of transformed cells.
  • Figure 16 is the result of the BrdU cell proliferation assay, and
  • Figure 17 is the result of the soft agar suspension independent growth assay of the cells. From Fig. 15 to Fig. 17, it was found that the cancerous characteristics and growth proliferation of the cells were inhibited after the DHX33 deletion.
  • Figure 18 After the deletion of DHX33 in lung adenocarcinoma cell line H1299 cells, the cell migration rate was significantly inhibited.
  • Example 5 Deletion of DHX33 reduces the ability of cells to form tumors in nude mice after transformation with c-Myc
  • This example was used to demonstrate the importance of DHX33 for c-Myc overexpression leading to cancer.
  • the cancer cells were treated with the lentivirus prepared in Example 1 to construct a stable cell line in which the DHX33 gene was silenced.
  • the cells were injected subcutaneously into immunodeficient mice and then monitored for tumor growth.
  • Cells containing higher c-Myc can form tumors in mice, but if the DHX33 is silenced to a substantially normal level, the cells greatly reduce the ability to form tumors in mice.
  • Figure 19 is the ability of cells to lose tumor formation in mice after knocking down DHX33.
  • Figure 20 is a quantitative analysis of tumor cells forming tumors in nude mice.
  • Example 6 Comparison of gene silencing efficiency of sh-DHX33-2 used in the present invention with a known small molecule RNA targeting DHX33 (sh-DHX33-1)
  • DHX33 regulation is one of the downstream pathways of the oncogene c-Myc.
  • DHX33 protein expression is high and DHX33 and c-Myc are co-positive in some cancer tissues (Fig. 13- - ie example 4).
  • Cell experiments confirmed that c-Myc can positively regulate the transcription of DHX33, and the decrease in the amount of DHX33 protein in cells transformed by c-Myc causes cell growth and carcinogenesis inhibition (Fig. 14-18---Example 5), This suggests that DHX33 plays a crucial role in the development and progression of cancer.
  • DHX33 is a potential target site for cancer therapy induced by c-Myc.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种靶向RNA解旋酶DHX33的小分子RNA或其互补序列、包含编码该小分子RNA或其互补序列的核酸序列的核酸以及含有所述小分子RNA或所述核酸序列的慢病毒质粒和慢病毒，所述小分子RNA包含序列TTGGGAAGCTGGTT GGCTATA，能够敲低DHX33的蛋白含量使之达到基本正常水平，降低了c-Myc的致癌性和维持癌性特征，从而达到治疗癌症的目的。

Description

一种靶向RNA解旋酶DHX33的小分子RNA及其应用
相关申请的交叉参考
本申请要求2017年1月23日提交的中国专利申请号201710049669.3、2017年1月23日提交的中国专利申请号201710049673.X、2017年1月23日提交的中国专利申请号201710049975.7、2017年1月23日提交的中国专利申请号201710049813.3的优先权权益，所述中国专利申请均通过引用而全文结合到本文中。
技术领域
本申请涉及生物技术领域，具体涉及一种靶向RNA解旋酶DHX33的小分子RNA、包含编码该小分子RNA或其互补序列的核酸序列的核酸、含有所述小分子RNA或所述核酸序列的慢病毒质粒和慢病毒，以及它们用于治疗癌症，尤其是肺腺癌的应用。
背景技术
c-Myc是在多种癌症中高表达的癌基因之一，可在多种癌症中出现基因扩增或易位(translocation)；同时，Myc的抑制蛋白Mga基因缺失突变也可引起Myc的过度表达。c-Myc在细胞内主要与Max结合，形成c-Myc/Max二聚体复合物的转录因子，发挥调控下游基因进而刺激细胞增生和代谢的功能，该功能的失调几乎出现在所有人类癌症中。针对癌细胞对癌基因的生长依赖(oncogene addiction)，向病人施用靶向不同癌蛋白的药物，可以专一性地抑制癌症的发展，延长病人寿命，实现精准个性化治疗。其中最突出的例子是EGFR的络氨酸激酶受体抑制剂EGFR-TKI，其已在临床上取得了很好的疗效。然而，尽管在小鼠模型中抑制c-Myc被证明能够有效遏制癌症的发生和发展，但由于c-Myc本身作为一个转录因子，是难以靶向的，至今医学上尚无有效的药物能针对性地抑制c-Myc信号途径的激活，由癌基因c-Myc扩增或激活引起的诸多癌症至今仍缺乏有效的治疗药物。
因此，针对c-Myc的上游或下游信号途径来筛选抗癌药物显得尤为重要。
发明内容
本申请的目的在于提供一种新的药物，该药物能够治疗由癌基因c-Myc扩增或激活引起的癌症。
在一个方面，本申请涉及一种靶向DHX33的小分子RNA或其互补序列，其特征在于所述小分子RNA包含以下的序列：
TTGGGAAGCTGGTTGGCTATA。
DHX33是RNA解旋酶家族的一个成员，参与了蛋白质翻译和核糖体RNA的生成，并且主动参与抑制细胞凋亡。DHX33是c-Myc下游的一个基因，在c-Myc诱导的癌症发生过程中起着至关重要的作用。所述小分子RNA能够敲低DHX33的蛋白含量，使之达到基本正常水平，降低c-Myc的致癌性和维持癌性特征，从而达到治疗癌症的目的。
在另一个方面，本申请涉及一种核酸，其包含编码所述小分子RNA或其互补序列的核酸序列，其特征在于所述核酸序列为以下的序列：
sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCC AACCAGCTTCCCAA-3’。
在另一个方面，本申请涉及包含所述小分子RNA或所述核酸序列的慢病毒质粒。
在另一个方面，本申请涉及包含所述小分子RNA或所述核酸序列的慢病毒。
根据本公开的以下描述并结合附图，本公开的这些和其他方面、特征和优点将变得显而易见。
附图说明
图1为本发明实施例1中蛋白含量的检测结果图。
图2为本发明实施例1中显微镜下观察细胞的凋亡情况的检测结果图。
图3为本发明实施例1中用流式细胞仪检测的细胞凋亡分析图。
图4为本发明实施例2中BT549蛋白含量的检测结果图。
图5为本发明实施例2中H1299蛋白含量的检测结果图。
图6为本发明实施例2中细胞凋亡基因Bcl-2和BAD信使RNA水平的检测结果图。
图7为本发明实施例2中分析DHX33结合在Bcl-2和BAD基因启动子的染色体免疫共沉降实验的PCR产物电泳结果图。
图8为本发明实施例3中DHX33mRNA量的实时荧光PCR检测结果图。
图9为本发明实施例3中DHX33蛋白量的检测结果图。
图10为本发明实施例3中c-Myc转录因子与启动子DNA结合的保守区域E-box中各个碱基示意图。
图11为本发明实施例3中DHX33基因近端启动子中含有的E-Box位点及序列的示意图。
图12为本发明实施例4中证明c-Myc蛋白直接结合在DHX33基因启动子产生电泳移动速率变化的分析结果。
图13为本发明实施例4中正常肺组织和肺癌组织实例中的DHX33的表达与c-Myc的表达对比图。
图14为本发明实施例4中各个蛋白量的分析图。
图15为本发明实施例4中细胞迁移实验结果。
图16为本发明实施例4中BrdU细胞增殖实验结果。
图17为本发明实施例4中细胞的软琼脂悬浮独立生长实验对比图。
图18为本发明实施例4中分析肺腺癌细胞中缺失DHX33后引起细胞迁移抑制的对比图。
图19为本发明实施例5中对照组与实验组在小鼠体内形成肿瘤的能力的对比图。
图20为本发明实施例5中对照组与实验组对裸鼠内形成肿瘤的定量分析的结果图。
图21为本发明实施例6中分析各种靶向DHX33的小RNA分子抑制DHX33蛋白表达的结果分析图。
具体实施方式
以下提供的每一个实施方案均有助于本公开某些方面的解释，但是不应解释为限制本公开的范围。而且，在整个说明书和权利要求书中，如本文中使用，由术语″约”修饰的值不限于指定的精确值，在一般情况，可对应于用于测量数值的仪器的精密度。
如本文使用，术语“治疗”包括限制、减缓、停止或逆转已有症状或病症的进展或严重性。
如本文使用，术语“患者”是指哺乳动物，如小鼠、豚鼠、大鼠、狗或人，优选患者为人。
本申请提供一种靶向RNA解旋酶DHX33的小分子RNA或其互补序列，所述小RNA分子为小发夹RNA，所述小RNA分子包含以下的序列：
TTGGGAAGCTGGTTGGCTATA。
在一个实施方案中，所述小RNA分子的序列为以下的序列：
TTGGGAAGCTGGTTGGCTATA(SEQ ID NO：17)。
发明人通过实验研究表明，在c-Myc引发的癌症的治疗上，DHX33是一个靶向位点，DHX33调控是癌基因c-Myc的下游途径之一，因此本发明的小分子RNA能够从癌基因c-Myc的下游信号途径来阻止癌症的发生和发展。所述小RNA分子具有敲低DHX33的蛋白含量使之达到基本正常水平的作用，其能够降低c-Myc的致癌性和维持癌性特征，从而达到治疗癌症的目的。
相应地，本申请还提供所述小分子RNA或其互补序列在制备用于治疗癌症的药物中的用途，其中所述癌症为由癌基因c-Myc扩增或激活引起的癌症。在一个实施方案中，所述癌症为肺腺癌。
本申请还提供一种核酸，其包含编码本发明的小分子RNA或其互补序列的核酸序列，所述核酸序列为以下的序列：
sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCC AACCAGCTTCCCAA-3’。
在一个实施方案中，所述核酸的序列为以下的序列：
sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCC AACCAGCTTCCCAA-3’。
本发明的核酸具有敲低DHX33的蛋白含量使之达到基本正常水平的作用，能够降低c-Myc的致癌性和维持癌性特征，从而达到治疗癌症，尤其是肺腺癌的目的。
相应地，本申请还提供本发明的核酸在制备用于治疗癌症的药物中的用途，其中所述癌症为由癌基因c-Myc扩增或激活引起的癌症。在一个实施方案中，所述癌症为肺腺癌。
可以通过现有慢病毒将本发明的核酸序列导入细胞内，以治疗由癌基因c-Myc扩增或激活引起的癌症，尤其是肺腺癌。
本申请还提供一种慢病毒质粒，其中所述慢病毒质粒包含所述小分子RNA。
本申请还提供一种慢病毒质粒，其中所述慢病毒质粒包含本发明的核酸序列：
sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGTATAG CCAACCAGCTTCCCAA-3’(SEQ ID NO：2)。
在一个实施方案中，本发明的慢病毒质粒中含有以下核酸序列：
sh-DHX33-2-前寡核苷酸：5’-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG 3’，
sh-DHX33-2-后寡核苷酸：5’-AATTCAAAAATTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3’。
本发明的慢病毒质粒含有编码靶向DHX33的小分子RNA序列或其互补序列的基因，所述小分子RNA具有敲低DHX33蛋白表达水平的作用。在本申请中，采用以下慢病毒质粒作为实验对照：
sh-DHX33-1：(5’-3’)：GCTATCGCAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGC(SEQ ID NO：1)，
其中sh-DHX33-1为以前公开的序列。
在本申请中，采用慢病毒载体pLKO.1-vector来构建慢病毒质粒，所述核酸序列通过限制酶切位点AgeI/EcoRI克隆到pLKO.1质粒中，需要说明的是本申请采用pLKO.1质粒仅仅是作为示例，本发明并不限于此。
为了构建sh-DHX33-1和sh-DHX33-2的慢病毒质粒，采用华大基因公司合成的DNA，它们分别是：
sh-DHX33-1-前寡核苷酸：5’-CCGGGCTATCGCAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGCTTTTTG 3’(SEQ ID NO：3)；
sh-DHX33-1-后寡核苷酸：5’一AATTCAAAAAGCTATCG CAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGC 3’(SEQ ID NO：4)；
sh-DHX33-2-前寡核苷酸：5’-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG-3’(SEQ ID NO：5)；
sh-DHX33-2-后寡核苷酸：5’-AATTCAAAAA TTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3’(SEQ ID NO：6)；
将上述序列克隆到pLKO.1质粒的限制酶切位点AgeI/EcoR中，即得到质粒pLKO.1-shRNA。
此外，本申请还提供本发明的慢病毒质粒在制备用于治疗癌症的药物中的用途，其中所 述癌症为由癌基因c-Myc扩增或激活引起的癌症。在一个实施方案中，所述癌症为肺腺癌。
本申请还提供一种慢病毒，所述慢病毒中含有本发明的慢病毒质粒。本发明的慢病毒具有敲低DHX33蛋白表达水平的作用，为一种基因靶向药物。
本发明的慢病毒可包含所述小分子RNA。或者，本发明的慢病毒包含编码所述小分子RNA或其互补序列的核酸序列：
sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCCAACCAGCTTCCCAA-3’。
在一个实施方案中，本发明的慢病毒含有以下核酸序列：
sh-DHX33-2-前寡核苷酸：5’-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG 3’，
sh-DHX33-2-后寡核苷酸：5’-AATTCAAAAATTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3’。
在一个实施方案中，所述慢病毒中的慢病毒质粒为pLKO.1质粒，所述编码所述小分子RNA或其互补序列的核酸序列通过限制酶切位点AgeI/EcoRI克隆到pLKO.1质粒中。
相应地，本申请还提供本发明的慢病毒在制备用于治疗癌症的药物中的用途，其中所述癌症为由癌基因c-Myc扩增或激活引起的癌症。在一个实施方案中，所述癌症为肺腺癌。
相应地，本申请还提供一种通过敲低DHX33的蛋白含量来治疗癌症的方法，所述癌症为由癌基因c-Myc扩增或激活引起的癌症。在一个实施方案中，通过将本发明的小分子RNA或其互补序列给予患者来治疗癌症。在另一个实施方案中，通过将本发明的核酸给予患者来治疗癌症。在一个实施方案中，通过将本发明的慢病毒质粒给予患者来治疗癌症。在另一个实施方案中，通过将本发明的慢病毒给予患者来治疗癌症。
本申请还提供一种制备本发明的慢病毒的方法，所述方法包括以下步骤：
培养293T细胞，待293T细胞生长到约90％的融合度时，与质粒混合物混合，其中所述质粒混合物包括慢病毒质粒；
经过16-18小时，更换成含有抗生素的培养基；再过24～48小时，收集细胞培养基，低速离心，收集上清液。
根据上述方法，所述质粒混合物可包括所述慢病毒质粒、包装辅助质粒pCMV-VSV-G和pCMV-dR8.2dvpr，混合的比例可以在宽范围内变化，优选约9∶8∶1的比例。转速可以在宽范围内变化，优选约1000～2000转/分钟，低速离心的时间为30秒至10分钟，优选约两分钟。抗生素为本领域中常用的任何抗生素，例如盘尼西林和/或链霉素。
例如，本发明的慢病毒可以通过以下方法来制备：
用Lipofectamine 2000(Life Technologies)脂质体导入技术将质粒混合物转染到293T细胞中。具体采用10厘米直径的细胞培养皿进行转染，待293T细胞生长到约90％的融合度时，将质粒pLKO.1-shRNA(即上述慢病毒质粒)、包装辅助质粒pCMV-VSV-G和pCMV-dR8.2dvpr混入其中，各质粒混合的比例为约9∶8∶1，使总DNA量达到每个培养皿12微克；
经过16-18小时，更换成含有抗生素(盘尼西林和链霉素)的培养基，再过约24或48小时，用无菌移液管收集细胞培养基，按1000～2000转/分钟的转速低速离心，时间约两分钟，将病毒分装于5毫升的无菌离心管中，存放于零下约80度的冰箱中。
本申请还提供一种细胞，其包含本发明的慢病毒质粒或本发明的慢病毒。
以下通过实施例作进一步说明，应当理解，此处所描述的具体实施例仅仅用于解释本发明，而不是用于限定本发明。
实施例1：癌细胞中缺失DHX33后迅速导致细胞凋亡
包括以下步骤：
1)根据目标小分子RNA序列，合成编码该目标小分子RNA序列的基因序列；该小分子RNA序列为靶向DHX33的小分子RNA序列；
2)将该基因序列克隆至病毒质粒中；
3)将病毒质粒转染到病毒载体上；
4)将病毒载体浸染乳腺癌细胞系BT549(HTB-122)和HCC1806(CRL-2335)；
5)浸染完成后，更换新培养基，进行传代培养；
6)培养一段时间后，提取总蛋白，用免疫印迹技术分析DHX33蛋白的表达量。
1、慢病毒质粒的构建：
实验组：靶向DHX33的小分子RNA的序列如下：
sh-DHX33-1：(5’-3’)：GCTATCGCAAAGTGATCATTT(作为阳性对照组)；
sh-DHX33-2：(5’-3’)：TTGGGAAGCTGGTTGGCTATA。
对照组：作为阴性对照的小分子RNA(shScrambled)序列为：
(5’-3’)：CCTAAGGTTAAGTCGCCCTCG。
将编码上述序列的核酸序列分别克隆到pLKO.1 vector的限制酶切位点AgeI/EcoRI上。
具体如下：
(1)将寡聚核酸用水溶解得到浓度为20微摩尔的溶液，将配对的前寡聚核酸和后寡聚核酸按以下的配方加入无菌PCR小管内：各5微升的前寡聚核酸，5微升的10XNEB缓冲液2(购买所得)和35微升的水。
(2)在100摄氏度处理10分钟。
(3)然后缓慢冷却到70摄氏度处理10分钟，然后再缓慢地冷却到室温，放置3个小时。
(4)将6微克的pLKO.1 TRC质粒(购买所得)加入5微升的10XNEB缓冲液1(购买所得)，1微升的限制内切酶AgeI，然后加水到50微升，混合好后在37摄氏度培育2个小时。用Qiaquick gel extraction kit(购买所得)提纯切割后的质粒片段，然后加入5微升的10XNEB缓冲液(针对EcoRI)，1微升的EcoRI，加水到50微升，在37摄氏度培育2个小时。
(5)用0.8％的低熔点琼脂糖凝胶分离得到的DNA片段，可以看到两个片段，切割7KB 的片段，然后提纯测定DNA浓度。
(6)用2微升前面准备好的退火后的寡聚核酸，加入20纳克的提纯的DNA片段，加入2微升10X的DNA连接酶缓冲液，和1微升的DNAT4连接酶，然后加水到20微升，放在室温过夜。
(7)第二天，用2微升的连接物转化50微升的XL-10gold大肠杆菌感受态细胞(购买所得)。将转化产物铺到含有每毫升培养基100微克氨苄青霉素的LB琼脂培养皿中，然后放置在37摄氏度过夜培育。
(8)第二天，用无菌牙签挑取单个菌落，置于含有上述浓度的氨苄青霉素的LB培养基中，在37摄氏度培育10个小时，然后用QIAGEN的质粒提取试剂盒提取质粒DNA。
(9)用500纳克的质粒DNA，向其中加入EcoRI/NcoI各0.5微升、2微升的10NEB缓冲液(针对EcoRI)，加水到20微升。在37摄氏度培育1个小时。
(10)用琼脂糖凝胶电泳筛选阳性克隆，阳性克隆有一个5KB和一个2KB的条带，测序分析鉴定核酸序列是否正确无误。
(11)扩增阳性克隆，然后用QIAGEN大抽质粒提取试剂盒提取高纯度质粒。
2、慢病毒的制备：用Lipofectamine 2000(Life Technologies)脂质体导入技术，将质粒混合物转染至293T细胞中。具体操作方法是采用10厘米的细胞培养皿做转染，待293T细胞生长到90％的融合度时将质粒pLKO.1-shRNA、包装辅助质粒pCMV-VSV-G和pCMV-dR8.2dvpr混合入其中，各质粒混合的比例为9∶8∶1，使总DNA量达到每个培养皿12微克。经过16-18小时，更换成含有抗生素盘尼西林和链霉素的培养基，再过24或48小时，用无菌移液管收集细胞培养基，低速离心，转速为2000转/分钟，时间为两分钟，将病毒分装于5毫升的无菌离心管中，存放于零下80度的冰箱中。
3、乳腺癌细胞系BT549和HCC1806从美国的ATCC获得。这两种细胞分别被包含编码小发夹RNA的核酸序列的慢病毒侵染，72小时后收集细胞并提取总蛋白。具体提取蛋白的方法是将细胞悬浮于细胞裂解液中(20毫摩尔的Tris-HCl，150毫摩尔的NaCl，1毫摩尔的EDTA，1％Triton-X-100，1％SDS，并补加蛋白酶和磷酸酶的抑制剂)。构建得到DHX33稳定缺失及稳定表达细胞系。用免疫印迹技术分析DHX33蛋白在各个样品中的表达量，用抗-GAPDH的抗体分析总蛋白上样量。
结果如图1所示，对照组DHX33稳定表达体系shSCR-BT549和shSCR-HCC1806，都能检测到DHX33蛋白；实验组DHX33稳定缺失体系1-sh-DHX33-BT549、1-sh-DHX33-HCC1806、2-sh-DHX33-BT549、2-sh-DHX33-HCC1806几乎检测不到DHX33蛋白。
4、为了分析癌细胞缺失DHX33后的表型，癌细胞中缺失了DHX33后，在显微镜下观察细胞的死亡现象。结果如图2所示，没有缺失DHX33的对照组细胞shSCR-BT549和shSCR-HCC1806生长旺盛，融合度高；而缺失DHX33的两组细胞1-sh-DHX33-BT549、 1-sh-DHX33-HCC1806、2-sh-DHX33-BT549、2-sh-DHX33-HCC1806表现出非常明显的死亡现象。说明了癌细胞中缺失了DHX33会迅速导致细胞死亡。
为进一步分析细胞在缺失DHX33之后是否有凋亡的现象将细胞进一步用Annexin V染色。具体方法是将细胞悬浮于磷酸缓冲液中(137毫摩尔的NaCl，2.7毫摩尔KCl，10毫摩尔的Na 2HPO 4，1.8毫摩尔KH 2PO 4)，使浓度达到每毫升1*10 6个细胞。然后在细胞的悬浮液中加入Annexin V-FITC，室温处理15分钟后，将细胞用70微米的滤网过滤，然后进行流式细胞仪的分析。结果如图3显示，细胞在缺失DHX33后有明显的细胞凋亡现象，对照组的凋亡比例很小。
实施例2：DHX33调控了控制细胞凋亡的重要基因Bcl-2和BAD
细胞凋亡是细胞在感受外界或内在环境的变化启动的程序性死亡过程。在由内因引起的细胞凋亡过程中，线粒体蛋白起了很重要的作用。线粒体内膜上的Bak/Bax形成聚合体通道，释放线粒体内的细胞色素c到细胞质中，随之引发了凋亡小体的生成和caspase的激活，进而引起不可逆的一系列蛋白酶切反应和细胞的凋亡。有两个蛋白调控Bak/Bax的聚合或激活，Bcl-2可以抑制Bak/Bax的激活，而BAD却抑制Bcl-2的活力。癌细胞一般有较高含量的Bcl-2蛋白或较低水平的BAD，从而抑制其凋亡。
1、为了分析DHX33对凋亡的抑制作用，在本实验中，选取了两种生长旺盛的癌细胞：肺腺癌细胞系H1299(CRL-5803)和乳腺癌细胞系BT549(HTB-122)，它们从美国的ATCC获得。这两种细胞分别被包含编码小发夹RNA的核酸序列的慢病毒(这里采用实施例中制备得到的克隆有sh-DHX33-1或sh-DHX33-2的慢病毒，以及对照组shScrambled)处理，96小时后收集细胞并提取总蛋白。总蛋白的提取方法如前所述。用免疫印迹技术分析细胞凋亡重要因子BAD和Bcl-2在各个样品中的表达量。
本实施例中同时也分析了DHX33的缺失效率和反应细胞凋亡的标志物：切割的PARP，用抗-微管蛋白的抗体分析总蛋白上样量。结果如图4和图5所示，对照组shSCR-BT549中，BAD的表达量低，shSCR-H1299中，Bcl-2表达量高；而缺失DHX33的1-sh-DHX33-BT549和2-sh-DHX33-H1299中，BAD的表达量高，而1-sh-DHX33-H1299和2-sh-DHX33-BT549中，Bcl-2表达量低。说明了DHX33对控制细胞凋亡的基因BAD和BCL-2有调控作用。
2、我们同时分析了上述细胞在缺失DHX33后BAD和Bcl-2信使RNA的表达水平。本实验中我们首先用Clontech试剂盒(Nucleospin RNA II)提取了细胞中的总RNA。用反转录酶将信使RNA反转录成为与其对应的互补DNA。然后通过定量PCR的方法分析Bcl-2和BAD信使RNA在各个样品中的含量。我们选择GAPDH作为内参，同时分析DHX33的信使RNA含量，以确定DHX33是否已经缺失。
采用的PCR引物如下：
人Bcl-2(前引物)-5′-ACAGTCCCATCAAAACTCCTG-3′(SEQ ID NO：7)；
人Bcl-2(后引物)-5′-TTACAGGCACAGAACATCCAG-3′(SEQ ID NO：8)；
人BAD(前引物)-5′-GACCTTCGCTCCACATCC-3′(SEQ ID NO：9)；
人BAD(后引物)-5′-AGTACTTCCGCCCATATTCAAG-3′(SEQ ID NO：10)。
结果如图6所示，在信使RNA水平，DHX33的降低导致这两个基因转录的变化；而且，克隆有sh-DHX33-2的慢病毒与克隆有sh-DHX33-1的慢病毒相比，其抑制DHX33表达的效果要更好。
3、为了研究这种调控的机制，我们更进一步分析DHX33蛋白是否直接调控了BAD和Bcl-2基因的转录。为此我们采用染色体免疫共沉降来分析DHX33是否结合在BAD和Bcl-2的启动子上。我们采用人肺腺癌细胞系H1299，将1*10 7细胞用胰蛋白酶消化之后，悬浮于完全的10毫升DMEM培养基中，加入37％甲醛，使浓度达到1％，混合使发生交联10分钟后，马上加入2.5摩尔甘氨酸，使浓度达到0.125摩尔，处理5分钟终止交联反应。然后每分钟1000转条件下，离心2分钟，去掉上清液，将细胞沉淀用磷酸缓冲液洗两次，离心方法同前。然后将细胞裂解，具体用2毫升的裂解液，裂解液含有1％SDS，50毫摩尔的Tris-HCl，2毫摩尔的EDTA，150毫摩尔的NaCl，和蛋白酶和磷酸酶的抑制剂。细胞在冰上裂解10分钟后，用40％输出的超声破碎仪充分裂解，剪短长链DNA使达到500bp-1000bp的大小。细胞裂解液经高速离心之后，提取上清液，然后用RIPA裂解液稀释五倍，分成均匀的三部分，加入50微升的蛋白A-琼脂糖珠在4摄氏度混合30分钟，然后离心获取上清液。分别加入抗体，包括anti-DHX33的抗体(Santa Cruz)，anti-TBP(Santa Cruz)和IgG作为阴性对照。在4摄氏度混合培育过夜，使抗体与抗原结合，然后加入蛋白A-琼脂糖25微升，使抗体结合蛋白A-琼脂糖，在4摄氏度混合1个半小时后，离心去掉上清液。将沉淀用含有0.5摩尔NaCl的RIPA缓冲液洗5次。然后用洗脱液(0.2摩尔的NaOH，1％SDS)洗脱免疫沉淀物，每次100微升，共洗2次。在洗脱下来的溶液中加入6摩尔的NaCl，使其浓度达到0.6摩尔浓度。然后在65摄氏度处理过夜，使DNA和蛋白解交联。用PCR产物提取试剂盒提取其中的DNA片段之后，用PCR的方法分析DHX33结合在BAD和Bcl-2启动子的效率。
分析BAD和Bcl-2启动子序列的引物序列：
人BAD启动子(前引物)-5′-CCACGCTCCTCTCTCCTAT-3′(SEQ ID NO：11)；
人BAD启动子(后引物)-5′-GGCTATGGGCGGAAGTTT-3′(SEQ ID NO：12)；
人Bcl-2(前引物)-5′-GGGAATCGATCTGGAAATCCTC-3′(SEQ ID NO：13)；
人Bcl-2(后引物)-5′-CCCATCAATCTTCAGCACTCT-3’(SEQ ID NO：14)。
结果如图7所示，DHX33直接作为转录因子调控这两个基因的转录。
实施例3：c-Myc可以正向调控DHX33的mRNA和蛋白表达
使用NIH3T3细胞(ATCC，ATCC-CRL-1658)，用编码c-Myc的慢病毒处理，用puromycin筛选得到稳定表达这一蛋白的细胞。96小时后，提取RNA及总蛋白，分析DHX33mRNA 及蛋白量，用总GAPDH做内参对照。结果如图8和图9所示，图8是DHX33mRNA量，图9是DHX33蛋白量。结果发现c-Myc处理的细胞有高含量的DHX33，当癌基因c-Myc在细胞中表达之后，细胞发生了癌性转化，在这一过程中，可以看到DHX33的蛋白出现迅速升高的现象。
图10：c-Myc癌基因作为转录因子识别特定的碱基序列，这些特定的碱基序列被命名为E-box，如图可见c-Myc转录因子与启动子DNA结合的保守区域E-box中各个碱基的保守型(Y-轴)。
图11：通过分析发现DHX33的近端启动子附近有多个E-box。如图所示，DHX33基因近端启动子中含有E-Box位点及序列。这些分析说明c-Myc可以直接结合在DHX33的启动子周围，调节DHX33的基因转录。
图12：EMSA实验分析c-Myc蛋白是否直接结合在DHX33的启动子上。
1)待标记探针的制备
克隆DHX33近端的2kb的启动子区域pGL3control质粒(购买所得)，然后用顶点诱变试剂盒(购买所得)将DHX33近端的3个E-box突变，得到野生型和突变型的DHX33启动子。分别以pGL3control DHX33野生型和pGL3control DHX33突变型为模板，以5’-CTAGCTAGCTAGTTTGGACAGAGAAGGGGAAAAC-3’(SEQ ID NO：15)和5’-CCGCTCGAGCGGCCCTCTCAGGTGCAGACAAC-3’(SEQ ID NO：16)为引物，PCR制备待标记探针，然后用PCR产物纯化试剂盒纯化。
2)生物素标记探针
(1)待标记探针95℃加热2分钟，立即冰浴使成单链；
(2)按如下方法37℃反应30分钟进行探针标记
水                     29μL
TdT buffer(5X)         10μL
待标记probe(1μM)      5μL
Biotin-11-dUTP(5μM)   5μL
TdT(10U/μL)           1μL
总体积                 50μL
(3)加入2.5微升标记终止液终止反应
(4)加入52.5微升氯仿-异戊醇(24：1)，涡旋，12000～14000g离心2分钟，上清液即为单链DNA探针
(5)加入退火缓冲液，加热至95℃，缓慢降温至室温，制得生物素标记的EMSA探针
3)按如下方式进行EMSA检测
(1)配制4％的聚丙烯酰胺凝胶10毫升
TBE buffer(10X)             0.5ml
Water                       8.1ml
39：1(29：1)丙烯酰胺        1.0ml
80％甘油                    312.5μL
10％APS                     75μL
TEMED                       5μL
(2)制备如下各样品。加入标记好的探针前先混匀，室温放置10分钟，并让冷探针优先反应，然后加入标记好的探针，混匀，室温放置20分钟。加入1μL上样缓冲液(10X，无色)，混匀立即上样。检测结果如下表1所示，各物质均采用微升为计量单位。
表1
Figure PCTCN2018072441-appb-000001
实施例4
为了进一步分析癌症组织中DHX33的表达与癌蛋白c-Myc表达的相关性，从而表明c-Myc可能是DHX33的上游调控基因，在肺癌组织中共染了c-Myc和DHX33，图13显示的是在很多肺癌组织中DHX33的表达与c-Myc的表达成正相关。图13在c-Myc阳性的肺癌组织中，DHX33蛋白同时显示阳性的比率大约66％(10/15)，图中第一行normal tissue是正常肺组织，图中第二～四行lung cancer是三个肺癌组织实例显示。
为了表明DHX33对c-Myc的致癌性和维持癌性特征至关重要，在c-Myc过表达的细胞体系中敲低DHX33的蛋白含量使之达到基本正常水平，观察在DHX33缺失后，c-Myc过表达的细胞的分裂生长是否会受到抑制。具体操作过程为：在NIH3T3细胞(ATCC，ATCC-CRL-1658)中，用c-Myc过表达慢病毒处理细胞，然后在c-Myc过表达的情况下分别用编码shSCR对照(shScrambled)和shDHX33(sh-DHX33-2)的慢病毒处理，使DHX33达到接近基本正常水平。图14的是各个蛋白量的分析图。图15是分析转化细胞的迁移率的实验结果。图16是BrdU细胞增殖实验结果，图17是细胞的软琼脂悬浮独立生长实验结果。从图15到图17中，可以发现在DHX33缺失后，细胞的癌性特征和生长增殖受到抑制。图18：在肺腺癌细胞H1299细胞中缺失DHX33后，可见细胞的迁移率发生了明显的抑制。DAPI标记的是细胞的迁移率在shSCR图有较高的蓝色标记细胞，而在shDHX33样品中几乎看不到细胞迁移，有很少的蓝色标记细胞。右面的明场图是所有被分析的细胞数，在对照组和实验组是几乎一样的。
实施例5：DHX33的缺失使细胞降低了被c-Myc转化后在裸鼠内成瘤的能力
本实施例用于证明DHX33对c-Myc过表达引起癌症发生的重要性。用实施例1中制备得到的慢病毒处理癌细胞，构建DHX33基因沉默的稳定细胞系。将细胞皮下注射入免疫缺陷小鼠体内，然后监测肿瘤的生长情况。含有较高c-Myc的细胞在小鼠体内可以形成肿瘤，但如果在沉默DHX33到基本正常水平后，细胞大大降低了在小鼠内形成肿瘤的能力。图19是细胞在敲低DHX33后失去在小鼠体内形成肿瘤的能力。图20是对肿瘤细胞在裸鼠内形成肿瘤的定量分析。
实施例6：比较本发明中所用的sh-DHX33-2与一种已知的靶向DHX33的小分子RNA(sh-DHX33-1)的基因沉默效率
可见DHX33蛋白的敲低效率非常明显，sh-DHX33-2的基因沉默效率显著优于sh-DHX33-1的基因沉默效率。
综上，研究表明DHX33调控是癌基因c-Myc的下游途径之一，在部分非小细胞肺癌中，DHX33蛋白表达很高且在部分癌组织中DHX33与c-Myc呈现共阳性(图13---即实施例4)。细胞实验证实c-Myc可以正向调控DHX33的转录，在被c-Myc转化的细胞中DHX33蛋白量下降会引起细胞生长和致癌性的抑制(图14-18---即实施例5)，这说明DHX33在癌症的发生和发展中起至关重要的作用，DHX33是c-Myc引发的癌症治疗上的一个潜在靶向位点。
本申请试图从c-Myc调控DHX33的角度出发研究DHX33对癌症发生发展的重要性。在分子机制上，我们发现DHX33解旋酶促进细胞生长的全部功能并不仅仅体现在促进核糖体生成和蛋白翻译上，我们的研究进一步揭示DHX33调控很多重要的细胞凋亡因子，这为攻克癌症提供了新通路和关键分子。
应当理解的是，本发明并不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (26)

  1. 一种小分子RNA或其互补序列，其特征在于所述小分子RNA包含以下的序列：
    TTGGGAAGCTGGTTGGCTATA。
  2. 权利要求1的小分子RNA或其互补序列，其特征在于所述小RNA分子的序列为以下的序列：
    TTGGGAAGCTGGTTGGCTATA。
  3. 权利要求1或2的小分子RNA或其互补序列在制备用于治疗癌症的药物中的用途，其特征在于所述癌症为由癌基因c-Myc扩增或激活引起的癌症。
  4. 权利要求3的用途，其特征在于所述癌症为肺腺癌。
  5. 一种核酸，其包含编码权利要求1或2的小分子RNA或其互补序列的核酸序列，其特征在于所述核酸序列为以下的序列：
    sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCCAACCAGCTTCCCAA-3′。
  6. 权利要求5的核酸，其特征在于所述核酸的序列为以下的序列：
    sh-DHX33-2：5’-TTGGGAAGCTGGTTGGCTATACTCGAGT ATAGCCAACCAGCTTCCCAA-3′。
  7. 权利要求5或6的核酸在制备用于治疗癌症的药物中的用途，其特征在于所述癌症为由癌基因c-Myc扩增或激活引起的癌症。
  8. 权利要求7的用途，其特征在于所述癌症为肺腺癌。
  9. 一种慢病毒质粒，其特征在于所述慢病毒质粒中含有权利要求1或2的小分子RNA或权利要求5或6的核酸序列。
  10. 权利要求9的慢病毒质粒，其特征在于所述慢病毒质粒中含有以下核酸序列：
    sh-DHX33-2-前寡核苷酸：5’-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG 3’，
    sh-DHX33-2-后寡核苷酸：5’-AATTCAAAAATTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3’。
  11. 权利要求9或10的慢病毒质粒，其特征在于所述慢病毒质粒为pLKO.1质粒，所述核酸序列通过限制酶切位点AgeI/EcoRI克隆到pLKO.1质粒中。
  12. 权利要求9至11中任一项的慢病毒质粒在制备用于治疗癌症的药物中的用途，其特征在于所述癌症为由癌基因c-Myc扩增或激活引起的癌症。
  13. 权利要求12的用途，其特征在于所述癌症为肺腺癌。
  14. 一种慢病毒，其特征在于所述慢病毒包含权利要求1或2的小分子RNA或权利要求5或6的核酸序列。
  15. 权利要求14的慢病毒，其特征在于所述慢病毒中含有以下核酸序列：
    sh-DHX33-2-前寡核苷酸：5’-CCGGTTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAATTTTTG 3’，
    sh-DHX33-2-后寡核苷酸：5’-AATTCAAAAATTGGGAAGCTGGTTGGCTATACTCGAGTATAGCCAACCAGCTTCCCAA-3’。
  16. 权利要求14或15的慢病毒，其特征在于所述慢病毒中的所述慢病毒质粒为pLKO.1质粒，所述核酸序列通过限制酶切位点AgeI/EcoRI克隆到pLKO.1质粒中。
  17. 权利要求14至16中任一项的慢病毒在制备用于治疗癌症的药物中的用途，其特征在于所述癌症为由癌基因c-Myc扩增或激活引起的癌症。
  18. 权利要求17的用途，其特征在于所述癌症为肺腺癌。
  19. 一种制备权利要求14至16中任一项的慢病毒的方法，所述方法包括以下步骤：
    培养293T细胞，待293T细胞生长到约90％的融合度时，与质粒混合物混合，其中所述质粒混合物包括所述慢病毒质粒；
    经过16-18小时，更换成含有抗生素的培养基；再过24～48小时，收集细胞培养基，低速离心，收集上清液。
  20. 权利要求19的方法，其特征在于所述质粒混合物包括按9∶8∶1的比例混合的所述慢病毒质粒以及包装辅助质粒pCMV-VSV-G和pCMV-dR8.2 dvpr。
  21. 权利要求19或20的方法，其特征在于低速离心进行约两分钟，转速为1000～2000转/分钟。
  22. 权利要求19至21中任一项的方法，其特征在于所述抗生素为盘尼西林和链霉素。
  23. 一种通过敲低DHX33的蛋白含量来治疗癌症的方法，所述癌症为由癌基因c-Myc扩增或激活引起的癌症。
  24. 权利要求23的方法，其中所述癌症为肺腺癌。
  25. 权利要求23或24的方法，所述方法包括给予患者权利要求1或2的小分子RNA或其互补序列，或者给予患者权利要求5或6的核酸，或者给予患者权利要求9至11中任一项的慢病毒质粒，或者给予患者权利要求14至16中任一项的慢病毒。
  26. 一种细胞，其特征在于所述细胞包含权利要求9至11中任一项的慢病毒质粒，或权利要求14至16中任一项的慢病毒。
PCT/CN2018/072441 2017-01-23 2018-01-12 一种靶向rna解旋酶dhx33的小分子rna及其应用 WO2018133733A1 (zh)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN201710049813.3 2017-01-23
CN201710049673.XA CN108342415B (zh) 2017-01-23 2017-01-23 一种用于治疗癌症的慢病毒质粒及其应用
CN201710049813.3A CN108339126B (zh) 2017-01-23 2017-01-23 一种用于治疗癌症的基因靶向药物及其应用
CN201710049669.3A CN108338986B (zh) 2017-01-23 2017-01-23 一种用于治疗癌症的小分子rna及其应用
CN201710049673.X 2017-01-23
CN201710049669.3 2017-01-23
CN201710049975.7A CN108342365B (zh) 2017-01-23 2017-01-23 一种用于治疗癌症的慢病毒及其制备方法应用
CN201710049975.7 2017-01-23

Publications (1)

Publication Number Publication Date
WO2018133733A1 true WO2018133733A1 (zh) 2018-07-26

Family

ID=62908937

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/072441 WO2018133733A1 (zh) 2017-01-23 2018-01-12 一种靶向rna解旋酶dhx33的小分子rna及其应用

Country Status (1)

Country Link
WO (1) WO2018133733A1 (zh)

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
FU, J. J. ET AL.: "Role of DHX33 in c-Myc-Induced Cancers", CARCINOGENESIS, vol. 38, no. 6, 5 November 2017 (2017-11-05), pages 649 - 660, XP055508224 *
LIU, Y. ET AL.: "The Interaction between the Helicase DHX33 and IPS-1 as a Novel Pathway to Sense Double-stranded RNA and RNA Viruses in Myeloid Dendritic Cells", CELLULAR & MOLECULAR IMMUNOLOGY, vol. 11, 16 September 2013 (2013-09-16), pages 50, XP055508239 *
MITOMA, H. ET AL.: "The DHX33 RNA Helicase Senses Cytosolic RNA and Activates the NLRP3 Inflammasome", IMMUNITY, vol. 39, 25 July 2013 (2013-07-25), XP055508236 *
ROBERT, F. ET AL.: "Perturbations of RNA Helicases in Cancer", WIRES RNA, vol. 4, 31 August 2013 (2013-08-31), pages 333 - 349 *
TIAN, Q. H. ET AL.: "DHX33 Expression is Increased in Hepatocellular Carcinoma and Indicates Poor Prognosis", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 473, 9 April 2016 (2016-04-09), pages 1163 - 1169, XP055508247 *
WEI, YUE ET AL.: "Adenovirus-Transferred Antisense c-myc Selectively Induces Tumor Cell Cycle Arrest and Apoptosis", NATIONNAL MEDICAL JOURNAL OF CHINA, vol. 79, no. 8, 31 August 1999 (1999-08-31), pages 617 - 620 *
WOOD, M. A. ET AL.: "An ATPase/Helicase Complex Is an Essential Cofactor for Oncogenic Transformation by c-Myc", MOLECULAR CELL, vol. 5, 29 February 2000 (2000-02-29), pages 321 - 330, XP002181332 *
YUAN, B. L. ET AL.: "DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle", MOLECULAR AND CELLULAR BIOLOGY, vol. 36, no. 23, 31 December 2016 (2016-12-31), XP055508234 *
ZHANG, Y. D. ET AL.: "p19ARF and RasV12 Offer Opposing Regulation of DHX33 Translation To Dictate Tumor Cell Fate", MOLECULAR AND CELLULAR BIOLOGY, vol. 33, no. 8, 11 February 2013 (2013-02-11), pages 1594 - 1607, XP055508261 *

Similar Documents

Publication Publication Date Title
US11332540B2 (en) Use of Circ-CDH1 inhibitors
JP2020534354A (ja) トリプルネガティブ乳癌の治療方法
EP4124347A1 (en) Circular rna molecule and use thereof in targeted degradation of protein of interest
CN108342365B (zh) 一种用于治疗癌症的慢病毒及其制备方法应用
JP2023181361A (ja) 過増殖性疾患を治療するためのｐｃｂｐ１の使用
CN109321656B (zh) 蛋白depdc1作为诊断三阴乳腺癌的标记物的用途
WO2018224044A1 (en) Use of upstream opening reading frame 45aa-uorf nucleotide sequence of pten gene and polypeptide coded by 45aa-uorf
CN108339126B (zh) 一种用于治疗癌症的基因靶向药物及其应用
US20140128450A1 (en) Cancer Therapy
WO2018133733A1 (zh) 一种靶向rna解旋酶dhx33的小分子rna及其应用
CN113230404A (zh) Sage1抑制剂在制备药物或试剂盒中的用途
AU2011256098A1 (en) Method for reducing expression of downregulated in renal cell carcinoma in malignant gliomas
JP2024506040A (ja) Ａｑｐ１ ＲＮＡを標的とするｓｇＲＮＡ及びそのベクターと使用
CN108338986B (zh) 一种用于治疗癌症的小分子rna及其应用
KR20090060212A (ko) 암의 검출 및 치료를 위한 조성물 및 방법
Qian et al. MiR-218-5p promotes breast cancer progression via LRIG1
JP6839707B2 (ja) Ｇｐｒ１６０を過剰発現する癌の予防、診断および治療
CN108342415B (zh) 一种用于治疗癌症的慢病毒质粒及其应用
JP5180084B2 (ja) がん細胞識別マーカー及びがん細胞増殖阻害剤
WO2023082242A1 (zh) Ctd-2256p15.2及其编码微肽作为靶点在开发肿瘤治疗药物中的应用
US9567583B2 (en) Method for treating glioma using Tarbp2 expression inhibitor
KR101817386B1 (ko) Elk3 단백질을 이용한 암 치료 또는 암 전이 억제제 스크리닝 방법 및 상기 elk3 단백질의 발현 또는 활성 억제제를 포함하는 암 치료 또는 암 전이 억제용 약학적 조성물
Betzler et al. BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression. Cancers 2023, 15, 310
CN115212308B (zh) Gasdermin e通路的靶向剂在治疗胰腺癌中的应用
US20230026844A1 (en) Inhibitors of Cancer Biomarkers and Uses Thereof

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: 18741586

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18741586

Country of ref document: EP

Kind code of ref document: A1