WO2023082242A1 - 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 - Google Patents

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 Download PDF

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WO2023082242A1
WO2023082242A1 PCT/CN2021/130604 CN2021130604W WO2023082242A1 WO 2023082242 A1 WO2023082242 A1 WO 2023082242A1 CN 2021130604 W CN2021130604 W CN 2021130604W WO 2023082242 A1 WO2023082242 A1 WO 2023082242A1
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tumor
ctd
patients
cells
chemotherapy
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French (fr)
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唐铁山
郭彩霞
周博
张传超
姚富文
谷峰
马勇杰
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中国科学院动物研究所
中国科学院北京基因组研究所(国家生物信息中心)
天津市肿瘤医院（天津医科大学肿瘤医院）
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to the application of CTD-2256P15.2 and its coded micropeptide as a target in the development of tumor treatment drugs.
  • DNA damage reagents are often used in tumor therapy, such as radiotherapy and many chemotherapy drugs, which mainly induce DNA damage in cancer cells, inhibit cancer cell proliferation, and induce their death.
  • tumor cells can often resist the effects of radiotherapy and chemotherapy drugs by changing their own DNA damage repair function, resulting in tumor drug resistance (including primary and secondary drug resistance).
  • PARP inhibitor (PARPi) is the first drug that successfully utilizes the concept of synthetic lethality to be approved for clinical use, and has achieved good therapeutic effects in the treatment of tumor patients with BRCA1/2 mutations.
  • PARPi can inhibit the catalytic activity of poly-ADP polymerases such as PARP1 and the repair of single-strand breaks, leading to the accumulation of DNA double-strand breaks (DSBs), or capture PARP1/2 on DNA strands, and interfere with their migration from the damaged site in time. This leads to replication stress and DSB damage.
  • PARPi is administered to tumor patients carrying BRCA1 or BRCA2 mutations, the DSB cannot be effectively repaired due to the inhibition of the DNA homologous recombination repair pathway, resulting in cell death.
  • Long non-coding RNA is a kind of non-coding RNA with a length greater than 200nt in cells, which plays an important regulatory role in various physiological and pathological processes, and its abnormal expression is often closely related to the occurrence, development and prognosis of tumors.
  • lncRNAs can regulate DNA damage repair process, such as NORAD, DDSR1 and lnc BGL3.
  • NORAD NORAD
  • DDSR1 DNA damage repair process
  • lncRNAs can regulate DNA damage repair process
  • these studies have focused on lncRNA as a molecular scaffold to bind specific proteins to regulate DNA repair.
  • lncRNAs related to DNA damage repair and tumor chemotherapy sensitivity are still unknown. very little.
  • lncRNA can encode biologically active micropeptides, thereby regulating tumor growth, invasion and metastasis.
  • micropeptides encoded by lncRNAs regulate DNA damage repair and tumor chemotherapy resistance.
  • One object of the present invention is to provide the use of CTD-2256P15.2 inhibitors.
  • the present invention provides the use of an inhibitor having any of the functions in the following a-d in at least one of the following 1)-9):
  • the nucleotide sequence of the CTD-2256P15.2 gene is sequence 1 or the 138th-272nd of sequence 1 in the sequence listing.
  • the amino acid sequence of the micropeptide PACMP is sequence 8 in the sequence listing.
  • the above-mentioned tumor treatment product is a tumor treatment drug, and its functions include inhibiting the proliferation of tumor cells or inducing the death of tumor cells or inhibiting the growth of tumors.
  • the inhibitor of CTD-2256P15.2 or its encoded micropeptide PACMP is the only active ingredient or one of the active ingredients.
  • the tumors include but not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, head and neck cancer, bladder cancer, cervical cancer, diffuse large B cell lymphoma, esophageal cancer, glial cell tumor, pancreatic cancer, prostate cancer, melanoma, thymoma, endometrial cancer.
  • the inhibitor refers to a molecule that can inhibit CTD-2256P15.2 or its encoded micropeptide PACMP, and the inhibitory effect includes but is not limited to: inhibiting the transcription or translation of CTD-2256P15.2, promoting CTD -2256P15.2 or PACMP degradation, inhibits PACMP function.
  • the inhibitor of CTD-2256P15.2 or micropeptide PACMP can be siRNA, shRNA, antisense RNA, miRNA, gene knockout or knockdown CRISPR-related plasmids and viral vectors, antibodies, polypeptides, small molecular compounds.
  • the inhibitor is siRNA or shRNA targeting CTD-2256P15.2 or gRNA targeting the coding region of PACMP or a vector expressing each of the above RNAs;
  • the nucleotide sequence of the siRNA targeting CTD-2256P15.2RNA is sequence 2 or sequence 3 in the sequence listing;
  • the nucleotide sequence of the shRNA targeting CTD-2256P15.2RNA is sequence 4 or sequence 5 in the sequence listing;
  • the nucleotide sequence of the gRNA targeting the PACMP coding region is sequence 6.
  • siRNA and shRNA inhibiting CTD-2256P15.2 refers to targeting the RNA sequence of CTD-2256P15.2 to reduce its RNA level, including the above sequence and all siRNA and shRNA with similar functions.
  • the other anti-tumor substances are other anti-tumor drugs or reagents or instruments required by other anti-tumor treatment methods.
  • the other tumor treatment drugs or methods refer to tumor treatment drugs or methods that can cause damage to tumor cell DNA or induce replication stress, including but not limited to: anthracyclines, camptothecin, PARP inhibitors, ATR inhibitors, CDK4/6 inhibitors, radiation therapy.
  • antineoplastic drug is a chemotherapeutic drug.
  • the inhibitor of CTD-2256P15.2 or its encoded micropeptide PACMP and other tumor therapeutic drugs or therapeutic methods are administered independently, and the route of administration may be the same or different, and it can be administered before the course of other tumor therapeutic drugs or therapeutic methods , during and after the above inhibitors were applied independently.
  • Another object of the present invention is to provide a product, which includes the above-mentioned inhibitor, and other reagents or instruments required for tumor treatment drugs or other anti-tumor treatment methods;
  • the product has at least one of the following functions:
  • Another object of the present invention is to provide the use of substances for detecting the expression level of CTD-2256P15.2 in tumor tissue.
  • the present invention also provides the application of substances and data processing devices for detecting the expression level of CTD-2256P15.2 in tumor tissue, which is any one of 1)-2):
  • the data processing device has a built-in module; the module has the following functions shown in (a1) and (a2):
  • the prognosis status of the patients to be tested in the low expression group after chemotherapy is better or better than the patients to be tested in the high expression group;
  • the prognostic overall survival period of the test patients in the low expression group after chemotherapy is longer or longer than that of the test patients in the high expression group;
  • the prognosis overall survival rate of the test patients in the low expression group after chemotherapy is higher or candidate higher than that of the test patients in the high expression group;
  • the prognostic progression-free survival of the patients in the low expression group after chemotherapy is longer or candidate longer than that of the patients in the high expression group;
  • the prognostic progression-free survival rate of the patients in the low expression group after chemotherapy is higher or higher than that of the patients in the high expression group;
  • the sensitivity of the test patients in the low expression group to chemotherapy drugs is higher or higher than that of the test patients in the high expression group.
  • the present invention also provides a system for predicting the prognosis of a tumor patient after chemotherapy, including a substance for detecting the expression level of CTD-2256P15.2 in tumor tissue and the above-mentioned data processing device.
  • the above-mentioned substance for detecting the expression level of CTD-2256P15.2 in tumor tissue is a probe or primer that specifically binds or amplifies the CTD-2256P15.2.
  • chemotherapeutic drugs used in the above chemotherapy are tumor therapeutic drugs that can cause DNA damage, including but not limited to anthracyclines, camptothecin or PARP inhibitors.
  • CTD-2256P15.2 The expression of CTD-2256P15.2 gene is used as the only detection index or one of the effective detection indexes in the tumor patient's sensitivity to chemotherapeutic drugs or prognosis evaluation reagent.
  • the present invention also provides following method:
  • the present invention provides a method for treating or adjuvantly treating tumors, comprising the following steps: using the above-mentioned inhibitor alone to treat tumors.
  • the present invention provides a method for treating or adjuvantly treating tumors, which includes the following steps: the above-mentioned inhibitor is used in combination with other drugs for treating tumors to treat tumors.
  • the present invention provides a method for treating or adjuvantly treating tumors, comprising the following steps: using the above-mentioned inhibitors in combination with other tumor treatment methods to treat tumors.
  • the present invention provides a method for auxiliary assessment of the prognosis of tumor patients after chemotherapeutic drug treatment, comprising the following steps: detecting the expression level of CTD-2256P15.2 in tumor tissues of tumor patients;
  • the present invention provides a method for assessing or assisting in assessing the sensitivity of tumor patients to chemotherapy drugs, comprising the following steps: detecting the expression level of CTD-2256P15.2 in the tumor tissue of the tumor patient;
  • the above substances for detecting the expression level of CTD-2256P15.2 in tumor tissue are specifically the primers used in the fluorescent quantitative PCR of the embodiment.
  • the prognostic status is specifically reflected in disease progression-free survival and/or overall survival, specifically:
  • the overall survival after chemotherapy of tumor patients corresponding to tumor tissues with high CTD-2256P15.2 gene expression is shorter or shorter than that of tumor patients corresponding to tumor tissues with low CTD-2256P15.2 gene expression;
  • the progression-free survival after chemotherapy of tumor patients corresponding to tumor tissues with high CTD-2256P15.2 gene expression is shorter or less than that of tumor patients corresponding to tumor tissues with low CTD-2256P15.2 gene expression.
  • the above chemotherapy drugs are tumor treatment drugs that can cause DNA damage, including but not limited to anthracyclines, camptothecin or PARP inhibitors;
  • drug resistance is chemotherapeutic drug resistance.
  • CTD-2256P15.2 is highly expressed in a variety of tumor types and can be induced by a variety of DNA-damaging chemotherapy drugs.
  • CTD-2256P15.2 can encode a functional micropeptide PACMP.
  • PACMP binds to the substrate adapter protein KLHL15 of the ubiquitin ligase Cul3, and competitively inhibits the binding and ubiquitination degradation of the latter with the important factor CtIP in the homologous recombination repair pathway; on the other hand, it binds to chemotherapy and DNA damage.
  • the induced PAR chain binding promotes the amplification of PAR signal, and the two synergistically promote the growth and drug resistance of tumor cells. Therefore, CTD-2256P15.2 or its encoded micropeptide PACMP is a new target for inhibiting tumor growth and enhancing clinical therapeutic effects of tumors.
  • Figure 1 shows the high expression of CTD-2256P15.2 in epirubicin-resistant breast cancer cell line MCF7-EPI.
  • Figure 2 shows that the expression of CTD-2256P15.2 is negatively correlated with the prognosis of breast cancer patients.
  • Figure 3 shows that knocking down CTD-2256P15.2 can enhance the sensitivity of various tumor cells to epirubicin.
  • Figure 4 shows that knocking down CTD-2256P15.2 can increase the sensitivity of tumor cells to various treatment options (including chemotherapy, targeted therapy and radiotherapy).
  • FIG. 5 shows that CTD-2256P15.2 can encode micropeptides.
  • Figure 6 shows that CTD-2256P15.2 regulates the chemosensitivity of tumor cells through its encoded micropeptide.
  • Figure 7 shows that inhibiting CTD-2256P15.2 or its encoded micropeptide can significantly inhibit tumor growth and enhance chemotherapeutic drug sensitivity.
  • Figure 8 shows that inhibiting CTD-2256P15.2 or its encoded micropeptide can reduce the efficiency of homologous recombination repair and micro-homologous end joining repair, and reduce the level of CtIP protein and the level of PAR induced by DNA damage.
  • the overall survival (Overall Survival, OS) in the following examples is defined as the time from enrollment to death from any cause or last follow-up.
  • the overall survival rate in the following examples is defined as the probability that a patient is still alive at a certain time point after being followed up at a certain time point.
  • PFS progression-free survival
  • the disease progression-free survival rate in the following examples is defined as the probability that no disease progression will be observed at a certain time point after a patient is followed up at a certain time point.
  • RNA CTD-2256P15.2 was highly expressed in epirubicin-resistant tumor tissues.
  • the nucleotide sequence of the long non-coding RNA CTD-2256P15.2 is sequence 1 in the sequence listing.
  • the MCF7-EPI cells in the logarithmic phase (the epirubicin-resistant cells obtained by gradually increasing the epirubicin concentration (from 20nM to 500nM) during the MCF7 cell culture process) and the control cell MCF7 (ATCC Cat#HTB -22, RRID: CVCL_0031) were inoculated into 96-well plates, with 4000 cells per well. After culturing for 12 hours, different concentrations of epirubicin (EPI) were added to the culture system of each culture well. After continuing to cultivate for 24 hours, the medium was aspirated, and fresh medium containing 10% (volume percentage) CCK8 reagent (DOJINDO, Cat#CK04) was added, and incubated for 4 hours. Then, the absorbance value of each well at 465 nm was detected with a microplate reader, and the relative cell survival curve was drawn.
  • EPI epirubicin
  • RNA of MCF7-EPI cells and MCF7 cell line was extracted with Trizol reagent, the extracted RNA was reverse transcribed into cDNA with reverse transcriptase, and then the RNA level of CTD-2256P15.2 was detected by real-time fluorescent quantitative PCR.
  • the primers for real-time fluorescent quantitative PCR detection were CTD-2256P15.2: forward primer: GACTTCTGCATTTGGCTGGAAGG, reverse primer: CTAACTCAGGGTATCGGAACCGA; internal reference GAPDH: forward primer: GGAGCGAGATCCCTCCAAAAT, reverse primer: GGCTGTTGTCATACTTCTCATGG.
  • tumor tissues of 92 breast cancer patients who received epirubicin-based chemotherapy were collected (from Tianjin Medical University Cancer Hospital) Among them, 48 patients had no tumor progression after chemotherapy, and the prognosis was good, which was the good prognosis group; 44 patients had disease progression after chemotherapy, and the prognosis was poor, which was the poor prognosis group (Table 1 and Table 2).
  • Table 1 shows the results of tumor tissue expression in 44 cases of poor prognosis breast cancer patients
  • Table 2 shows the results of tumor tissue expression in 48 cases of breast cancer patients with good prognosis
  • RNA extraction kit was used to extract tumor tissue RNA.
  • the specific extraction steps refer to the instructions of the kit. Then the extracted RNA was reverse-transcribed into cDNA by reverse transcriptase, and then the expression of CTD-2256P15.2 in these tumor tissues was detected by real-time fluorescent quantitative PCR.
  • the method of fluorescent quantitative PCR technology is the same as the above-mentioned two real-time fluorescent quantitative PCR.
  • A is the expression level of CTD-2256P15.2 in patients with poor prognosis and good prognosis
  • B progression-free survival
  • C overall survival
  • detecting the expression level of CTD-2256P15.2 gene in tumor tissue can predict the sensitivity of tumor patients to chemotherapy drugs; or detecting the expression level of CTD-2256P15.2 gene in tumor tissue can predict the prognosis status of tumor patients, specifically reflected in Progression-free survival and/or overall survival;
  • Tumor patients corresponding to tumor tissues with high CTD-2256P15.2 gene expression are less sensitive to chemotherapy drugs than tumor patients corresponding to tumor tissues with low CTD-2256P15.2 gene expression;
  • Tumor patients with high CTD-2256P15.2 gene expression in tumor tissue have poorer prognosis after chemotherapy than tumor patients with low CTD-2256P15.2 gene expression in tumor tissue;
  • the overall survival period after chemotherapy of tumor patients corresponding to tumor tissues with high CTD-2256P15.2 gene expression is shorter than that of tumor patients corresponding to tumor tissues with low CTD-2256P15.2 gene expression;
  • the progression-free survival period after chemotherapy of tumor patients corresponding to tumor tissues with high CTD-2256P15.2 gene expression is shorter than that of tumor patients corresponding to tumor tissues with low CTD-2256P15.2 gene expression.
  • CTD-2256P15.2 Quantitative transcriptome sequencing data of various TCGA tumors were searched on the GePIA website.
  • the Ensembl ID of CTD-2256P15.2 gene is ENSG00000259802, and the expression of CTD-2256P15.2 in various tumor types can be obtained by searching the ID.
  • BLCA bladder urothelial carcinoma
  • BRCA invasive breast carcinoma
  • CESC cervical squamous and adenocarcinoma
  • COAD colon cancer
  • DLBC diffuse large B-cell lymphoma
  • ESCA esophageal carcinoma
  • LGG brain low-grade glioma
  • LUAD lung adenocarcinoma
  • LUSC lung squamous cell carcinoma
  • OV ovarian serous cystadenocarcinoma
  • PAAD pancreatic cancer
  • PRAD prostate cancer
  • READ rectal adenocarcinoma
  • STAD gastric cancer
  • THYM thymic carcinoma
  • UCEC endometrial carcinoma
  • UCS uterine sarcoma.
  • TPM Transcripts Per Million
  • CTD-2256P15.2 can be selected as a therapeutic target in multiple tumors for the following experiments.
  • siRNA targeting CTD-2256P15.2 In order to detect the regulatory effect of CTD-2256P15.2 on the chemosensitivity of tumor cells, two specific siRNA targeting CTD-2256P15.2 were designed and synthesized according to the RNA sequence of CTD-2256P15.2 (CTD-2256P15.2 inhibitory agent siRNA) silnc15.2-1 and silnc15.2-2, the sequence is:
  • silnc15.2-2 GCGGCUUCUGGAGGGACAA (sequence 2);
  • silnc15.2-1 GCAGAUGACCUAGCACAAA (SEQ ID NO: 3);
  • control siRNA is: UUCUCCGAACGUGUCACGU.
  • RNAiMAX Lipofectmine RNAiMAX (Invitrogen, Cat#13778150), the control siRNA (siNC), specific silnc15.2-1 and silnc15.2-2 targeting CTD-2256P15.2 were transfected into breast cancer cells (MCF7 ATCC Cat# HTB-22, RRID: CVCL_0031, MDA-MB-231 ATCC Cat#CRM-HTB-26, RRID: CVCL_0062), osteosarcoma cells (U2OS Cat#HTB-96; RRID: CVCL_0042), gastric cancer (AGS ATCC Cat#CRL -1739,RRID:CVCL_0139), cervical cancer cells (HeLa Cat#CRL-7923; RRID:CVCL_0030), ovarian cancer cells (SK-OV-3Cat#HTB-77; RRID:CVCL_0532) and non-small cell lung cancer cells (A549Cat# In CCL-185; RRID: CVCL_0023), the working concentration of
  • the CCK8 method detects the survival of different tumor cells transfected with CTD-2256P15.2 inhibitor siRNA.
  • the results are shown in Figure 3C-3I. It can be seen that compared with the control siRNA, CTD-2256P15.2 inhibitory siRNA knocks down different tumor cells CTD-2256P155.2 in the cells can significantly enhance the epirubicin sensitivity of these cells and reduce the survival rate of tumor cells; it indicates that CTD-2256P15.2 is an effective target to enhance the killing effect of tumor chemotherapy drugs and improve the clinical treatment effect.
  • siRNA that knocks down or inhibits the expression of CTD-2256P15.2 can enhance the sensitivity of tumor cells to chemotherapy drugs and reduce the survival rate of tumor cells after treatment with chemotherapy drugs.
  • siRNA synthetic control siRNA
  • siRNAiMAX lipofectmine RNAiMAX
  • the working concentration of siRNA was 100 nM.
  • CPT camptothecin
  • CTD-2256P15.2 inhibitory siRNA inhibited the expression of CTD-2256P15.2 can significantly increase the MCF7 cells against camptothecin (CPT), ATR inhibitor (VE-822) and CDK4/6 inhibitor (Palbociclib) these tumors Sensitivity to therapeutic drugs.
  • CTD-2256P15.2 inhibitory siRNA can significantly enhance the killing effect on tumor cells, providing a new strategy for improving the efficacy of tumor therapy.
  • CTD-2256P15.2 inhibitory siRNA can enhance the killing effect of antitumor drugs on tumor cells and reduce the chemotherapy resistance of tumor cells.
  • ShRNA technology was used to reduce the expression level of CTD-2256P15.2, and the survival of tumor cells after Olaparib or X-ray treatment was detected by colony formation assay. Specific steps are as follows:
  • shlnc15.2-2 GCGGCTTCTGGAGGGACAAAGCTCGAGCTTTGTCCCTCCAGAAGCCGC (sequence 4); shlnc15.2-1: GCAGATGACCTAGCACAAATACTCGAGTATTTGTGCTAGGTCATCTGC (sequence 5).
  • shNC sequence TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA
  • the recombined pLKO-shlnc15.2-2 plasmid is the carrier obtained between the AgeI and EcoRI sites of the pLKO vector by replacing the coding gene of shlnc15.2-2 shown in sequence 4, which expresses shlnc15.2-2 (sequence 4 coding RNA).
  • the recombined pLKO-shlnc15.2-1 plasmid is the carrier obtained between the AgeI and EcoRI sites of the pLKO vector by replacing the coding gene of shlnc15.2-1 shown in sequence 5, which expresses shlnc15.2-1 (sequence 5 coding RNA).
  • the recombinant pLKO-shNC plasmid is a vector obtained by replacing the coding gene of shNC between the AgeI and EcoRI sites of the pLKO vector.
  • 293T cells in logarithmic growth phase (Cat#CRL-3216; RRID: CVCL_0063) were inoculated in a 10 cm culture dish to make the cell density about 50%, and plasmid transfection was carried out after culturing overnight.
  • the medium Before transfection, the medium was replaced with fresh medium without antibiotics and placed at 37°C. According to the instructions of the vigoFect transfection reagent, 5 ⁇ g of the plasmid (the above-mentioned pLKO-shlnc15.2-1 or pLKO-shlnc15.2-2 and the viral packaging plasmid pRSV-Rev (Addgene ID 12253), pMD2.G (Addgene ID 12259), pMDLg/ pRRE (Addgene ID 12251) (ratio 2:1:1:1) was transfected into 293T cells. After 24 hours of transfection, the medium was discarded, and 8ml of fresh complete medium was added to continue the culture. After 48 hours, the cells were cultured The serum contains the corresponding lentivirus.
  • the plasmid the above-mentioned pLKO-shlnc15.2-1 or pLKO-shlnc15.2-2 and the viral packaging
  • MCF7 cells were inoculated into a 6cm culture dish one day in advance to make the density 50% for lentivirus infection.
  • the 293T culture supernatant was collected and filtered through a 0.45 ⁇ M filter into a clean centrifuge tube.
  • the filtered virus was mixed with an equal volume of fresh medium, and polybrene with a final concentration of 10 ⁇ g/mL was added, mixed well and added to MCF7 cells, and the medium was changed 8 hours after infection. After continuing to culture for 48 hours, add 1mg/ml puromycin for screening.
  • MCF7 shRNA-1 and MCF7 shRNA-2 stable expression targeting CTD-2256P15.2 shRNA(shCTD-2256P15.2)-1/2 MCF7 cells, named MCF7 shRNA-1 and MCF7 shRNA-2.
  • the cells transformed with the recombinant pLKO-shNC plasmid were used as the control, named MCF7 shNC.
  • the above-mentioned qPCR detection method is as follows: extract the total RNA of the above-mentioned cells, use reverse transcriptase to reverse-transcribe the RNA into cDNA, and detect the content of CTD-2256P15.2 by fluorescent quantitative PCR (primers are the same as before). The results are shown in Figure 4D. It is concluded that shlnc15.2-1 and shlnc15.2-2 can knock out CTD-2256P15.2 and reduce its expression.
  • MCF7 shRNA-1 or MCF7 shRNA-2 and MCF7 shNC obtained in the above 2 into 6 cm culture dishes respectively.
  • Example 3 the micropeptide encoded by CTD-2256P15.2 and its regulation of chemosensitivity of tumor cells
  • the open reading frame 1 (ORF1, 138-272 of sequence 1) of CTD-2256P15.2 may encode a 44-amino acid micropeptide, the amino acid sequence of which is MAASGGTKKA QSGGRRLREPSSRPSRRARQRPRRGALRKAGRFL (sequence 8 ).
  • CRISPR-Cas9 technology was used to knock in a sequence containing SBP-FLAG tag at the 3' end of ORF1 of CTD-2256P15.2 gene in U2OS cells (before the stop codon).
  • the monoclonal cells with successful insertion were obtained by picking the monoclonal cells and carrying out PCR sequencing identification.
  • the recombinant vector pX330-gRNA is a vector obtained by inserting the DNA molecule shown in CGGCGTGCACTGTCGGTCGGCGG (sequence 6) between the Bbs1 sites of the pX330 vector (Addgene ID 42230), which expresses gRNA (gRNA encoded by sequence 6).
  • the donor DNA is the left homology arm-tag-containing sequence (SBP-FLAG-P2A-puromycin)-right homology arm, and the nucleotide sequence is sequence 7.
  • the above donor DNA and pX330-gRNA plasmids were transferred into U2OS cells (ATCC, Cat#HTB-96; RRID: CVCL_0042) by electroporation using the Pulse Generator-CUY21EDDID 2 electroporation instrument with a voltage of 135V. After 48 hours, 1 mg/ml puromycin was added for selection, and then the cells grown after puromycin selection were monoclonal inoculated. After 2 weeks, the monoclonal cells were picked and expanded for culture, and then the genomic DNA was extracted, and PCR amplification and sequencing were used to identify the successful knock-in positive clone U2OS-KI cells.
  • the primers used in the above PCR amplification are: forward primer: AGAGGCTGACAGAAAGCGAG, reverse primer: CTAACTCAGGGTATCGGAACCGA, obtained a fragment of about 1580bp and sequenced a positive clone containing a knock-in tag sequence, named U2OS-KI cells, the cell Expression fusion protein ORF1-SBP-FLAG-P2A (the coding nucleic acid of this fusion protein is sequence 10, and it is that the 3' end (before stop codon) of the first open reading frame of CTD-2256P15.2 is fused to SBP-FLAG-P2A - the sequence obtained by puromycin, when the protein is translated, the P2A sequence is self-cleaved and broken, thereby obtaining the ORF1-SBP-FLAG-P2A fusion protein).
  • the shRNA technology knocks down CTD-2256P15.2 in the U2OS-KI cells obtained in the above 3), the method refers to the fourth part of Example 2, and the recombinant pLKO-shlnc15.2-1 plasmid is used to transfect U2OS-KI cells with lentivirus
  • the obtained cells were named U2OS-KI-shlnc15.2.
  • the cells obtained by transfecting U2OS-KI cells with the recombinant pLKO-shNC plasmid through lentivirus were named U2OS-KI-shNC.
  • ORF1 fusion protein The expression of ORF1 fusion protein was detected by microscope.
  • WT is U2OS cells
  • KI-shlnc15.2 is U2OS-KI-shlnc15.2 cells
  • KI-shNC is U2OS-KI-shNC cells
  • ORF1 can initiate translation and express ORF1-SBP-FLAG-P2A fusion protein, while there is no fusion protein expression in lnc15.2 knockdown cells U2OS-KI-shlnc15.2.
  • ORF1 can initiate translation and express ORF1-SBP-FLAG-P2A fusion protein, and ORF1-SBP in sh15.2 knockdown cells U2OS-KI-shlnc15.2 -
  • the expression of FLAG-P2A was significantly decreased compared to U2OS-KI-shNC cells.
  • ORF1 of CTD-2256P15.2 could encode a 44 amino acid long micropeptide.
  • CTD-2256P15.2 regulates the chemosensitivity of tumor cells through its encoded micropeptide
  • CTD-2256P15 which can resist shCTD-2256P15.2, was complemented in MCF7 and U2OS cells with stable knockdown of CTD-2256P15.2 .2 Wild-type full-length (FL) and ORF1 start codon mutated full-length (FL*), as well as complement ORF1 fusion protein, to detect the sensitivity of cells to epirubicin.
  • MCF7 and U2OS cells stably knocked down by CTD-2256P15.2 were constructed using shRNA technology, and the specific steps refer to Example 2. The difference is that the cells are MCF7 and U2OS cells, and the recombinant plasmid is the pLKO-shlnc15.2-1 plasmid, and the MCF7 cells stably expressing the shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15. shRNA to CTD-2256P15.2 (shCTD-2256P15.2)-1 U2OS cells.
  • the recombinant pLKO-shNC plasmid knockout was used as a control to obtain shNC-expressing MCF7 cells and shNC-expressing U2OS cells.
  • the recombinant plasmid FL is a vector obtained by cloning the gene sequence (sequence 1) of CTD-2256P15.2 into the Nhe1 and EcoR1 restriction sites of the pNL lentiviral expression vector;
  • Recombinant plasmid FL* is the ORF1 start codon mutation sequence of CTD-2256P15.2 (its sequence is the ATG mutation of the sequence shown in the 138-141 position of sequence 1 to ATT, that is, the start codon of the open reading frame No. 1 ATG is mutated to ATT), and then cloned into the vector obtained between the Nhe1 and EcoR1 restriction sites of the pNL lentiviral expression vector;
  • Recombinant plasmid ORF1 is the Nhe1 and EcoR1 restriction enzymes that clone the coding gene of ORF1-SBP-FLAG fusion protein (its sequence is sequence 9, that is, the 138th-269th position of sequence 1 + SBP-FLAG nucleic acid sequence) into the pNL lentiviral expression vector The vector obtained between the sites;
  • the above-mentioned cells were inoculated into a 96-well plate, and the adherent cells were treated with 1 ⁇ M epirubicin for 24 hours, and the survival rate of the cells was detected by CCK8 reagent, the method was the same as in Example 1.
  • MCF7 cells expressing shNC in control U2OS cells expressing shNC in control, MCF7 cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2)-1 and stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2)-1 U2OS cells were used as control.
  • A is MCF7 cells
  • B is U2OS cells
  • shNC indicates cells expressing shNC
  • shlnc15.2 indicates cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2)-1
  • shlnc15.2+ORF1 means cells where ORF1 complements shRNA(shCTD-2256P15.2)-1
  • shlnc15.2+FL means cells where FL complements shRNA(shCTD-2256P15.2)-1
  • knocking down CTD-2256P15.2 can increase the sensitivity of tumor cells to epirubicin, and complement the expression of wild-type full-length (ORF1) and ORF1 fusion protein (FL) could restore the reduced epirubicin sensitivity of the cells, whereas full-length complementation of the ORF1 mutation (FL*) could not.
  • a triple-negative breast cancer cell line MDA-MB-231 with stable knockdown of CTD-2256P15.2 and complementation of wild-type full-length or ORF1 mutant full-length was constructed, and a certain amount of cells were inoculated into the mammary fat pad of female nude mice Down.
  • the mice were treated with normal saline and epirubicin respectively, and the tumor size was measured and counted.
  • the mice were sacrificed and the tumors were dissected out, photographed and the tumor weight was measured.
  • Example 2 the recombinant pLKO-shlnc15.2-1 plasmid was transferred into the MDA-MB-231 cell line to obtain MDA- MB-231 cells;
  • control knockdown MDA-MB-231 cells expressing shNC were obtained by transfecting the recombinant pLKO-shNC plasmid as a control.
  • the wild-type lnc15.2 full-length (FL) and the mutant lnc15.2 full-length (FL*) were respectively transferred into stable expression targeting CTD-2256P15.2 shRNA (shCTD-2256P15.2) -1 of MDA-MB-231 cells, MDA-MB-231 cells of FL supplemented with shRNA(shCTD-2256P15.2)-1 and MDA-MB of FL* supplemented with shRNA(shCTD-2256P15.2)-1 -231 cells.
  • mice 4 ⁇ 10 6 of the above cells in the logarithmic growth phase were mixed with an equal volume of Matrigel, and inoculated under the mammary fat pad of female BALB/c nude mice.
  • the mice were randomly divided into two groups, which were injected intraperitoneally with 5 mg/kg epirubicin or an equal volume of normal saline. Administer once every 4-5 days, measure the tumor volume every other day, and draw the relative growth curve of the tumor. The mice were sacrificed on the 11th day after the initial administration, the tumors were dissected out, photographed and the tumor weight was measured.
  • MDA-MB-231 cells expressing shNC and MDA-MB-231 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 were used as controls.
  • A-C are respectively (A) the growth situation of each group of tumor cells transplanted with epirubicin treatment or no treatment, (B) the weight of each group of transplanted tumors collected, (C ) photographs of transplanted tumors collected in each group.
  • shNC means MDA-MB-231 cells expressing shNC
  • shlnc15.2 means MDA-MB-231 cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2)-1
  • shlnc15.2+FL means Complementation of wild-type full-length lnc15.2 in shlnc5.2MDA-MB-231 cells
  • shlnc15.2+FL* indicates complementation of mutant full-length lnc15.2 in shlnc5.2MDA-MB-231 cells, regardless of tumor origin volume, tumor or tumor size, it can be seen that knocking down CTD-2256P15.2 significantly inhibited tumor growth and increased sensitivity to epirubicin. Sensitivity to epirubicin.
  • siBRCA1 UUAAGACCUCUGGCAUGAAU
  • siCtIP GCUAAAACAGGAACGAAUC
  • the GFP-positive cells were sorted by flow cytometry, and the GFP-positive cells and the total cells were counted respectively.
  • the ratio of positive cells to the total cells was the DNA homologous recombination repair (HR) efficiency or microhomology-mediated terminal Link (MMEJ) efficiency.
  • MCF7 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 prepared above and MCF7 cells complementing shRNA (shCTD-2256P15.2)-1 by ORF1 (transformed into recombinant plasmid ORF1 ), Fl* supplemented with shRNA (shCTD-2256P15.2)-1 MCF7 cells (transformed into the recombinant plasmid FL*), the cells in the logarithmic growth phase were collected, and the protein level of CtIP in the cells was detected by western blot method.
  • the pNL lentiviral empty expression vector i.e. the pNL lentiviral expression vector
  • the recombinant plasmid FL and the recombinant plasmid FL* were respectively transferred into silnc15.2-1 knockdown MCF7 cells to obtain the pNL lentiviral empty expression vector to complement silnc15.2 -1 MCF7 cells, FL supplemented with silnc15.2-1 MCF7 cells and FL* supplemented with silnc15.2-1 MCF7 cells.
  • the above-mentioned cells in the logarithmic growth phase were seeded into a 6-well plate so that the cell density was about 70%. The next day, discard the medium, treat the cells with serum-free culture containing 400 ⁇ M H 2 O 2 at 37 degrees for 5 minutes, discard the medium, wash the cells twice with PBS, and add 1 ⁇ SDS sample buffer to lyse the cells. The level of intracellular PAR was detected by western blot. Serum-free medium without H 2 O 2 was used as a control.
  • siNC is the MCF7 cells treated with siNC
  • silnc15.2 is the MCF7 cells treated with silnc15.2-1
  • - is the MCF7 cells with pNL lentiviral empty expression vector complemented with silnc15.2-1
  • FL is FL complements silnc15.2-1 MCF7 cells
  • FL* is FL* supplements silnc15.2-1 MCF7 cells
  • CtIP is a key factor in the normal initiation of DNA homologous recombination repair and end link repair mediated by microhomology, and the two DNA damage repair pathways were impaired after CtIP deletion.
  • Simultaneous downregulation of CtIP and/or PAR by inhibiting CTD-2256P15.2 or its encoded micropeptide PACMP can inhibit tumor growth through synthetic lethal effects.
  • the inhibitor of CTD-2256P15.2 or the micropeptide PACMP encoded by the present invention acts on tumor cells or tumor tissues, it can significantly inhibit the growth of tumor cells, increase the apoptosis of tumor cells, and reduce the tumor volume. antitumor effect.
  • the new anti-tumor drug combination scheme provided by the present invention uses CTD-2256P15.2 or its encoded micropeptide PACMP inhibitor in combination with other anti-tumor drugs, which can significantly enhance the killing effect of anti-tumor drugs on tumor cells and reduce the risk of tumor cells. Chemotherapy resistance, thereby improving the clinical treatment effect of tumors.
  • CTD-2256P15.2 is highly expressed in chemotherapy-resistant tumor tissues and cell lines, and its high expression is significantly negatively correlated with disease progression-free survival and overall survival of tumor patients.
  • the CTD2256P15.2 gene expression level provided by the present invention can be used as a molecular index for predicting chemotherapy sensitivity and prognosis of tumor patients, creating a new standard for effectively guiding clinical chemotherapy drugs for tumor patients and evaluating treatment prognosis.

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Abstract

本发明提供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。本发明还提供CTD-2256P15.2抑制剂的如下用途：1)制备肿瘤治疗产品；2)制备降低肿瘤细胞对化疗药物的耐药性的产品；3)制备提高肿瘤细胞对化疗药物的敏感性的产品。

Description

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 技术领域
本发明属于生物技术领域，尤其涉及一种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。
背景技术
DNA损伤试剂常被用于肿瘤治疗，如放射治疗及许多化疗药物，这主要是通过在癌细胞中诱发DNA损伤，抑制癌细胞增殖、诱发其死亡。然而肿瘤细胞往往能通过改变自身的DNA损伤修复功能来抵抗放疗和化疗药物的作用，产生肿瘤耐药性(包括原发和继发耐药)。PARP抑制剂(PARPi)是第一款成功利用合成致死概念获批临床使用的药物，在治疗BRCA1/2突变的肿瘤患者中取得了很好的治疗效果。目前认为，PARPi能抑制PARP1等聚ADP聚合酶的催化活性及单链断裂修复，导致DNA双链断裂(DSB)积累，或者捕获DNA链上的PARP1/2，干扰其从损伤位点的及时移除，进而引发复制压力和DSB损伤。携带BRCA1或BRCA2突变的肿瘤患者给予PARPi后，由于DNA同源重组修复通路受抑制，DSB不能被有效修复，进而导致细胞死亡。然而，与临床上广泛使用的化疗药物相似，PARPi的使用同样也导致耐药性产生，其中很大程度上是由于DNA损伤修复功能回复引发的，这些耐药性的产生严重制约着PARPi的临床使用和疗效。目前已知超过40％的BRCA突变卵巢癌患者不能从PAPRi治疗中获益。因此找到调控DNA损伤应答、化疗药物和PARPi耐药的关键分子，不仅有助于发现预后生物标志物，还有助于发现新靶点，开发组合疗法，提高肿瘤治疗疗效。
长链非编码RNA(lncRNA)是细胞内一类长度大于200nt的非编码RNA，在多种生理、病理过程中发挥重要调控作用，其异常表达常与肿瘤发生、发展和预后密切相关。研究发现一些lncRNA能调控DNA损伤修复过程，例如NORAD，DDSR1和lnc BGL3。然而，这些研究都集中在lncRNA作为分子支架结合特定蛋白来调控DNA修复方面，相对于lncRNA多样的作用机制和重要的调控功能而言，对DNA损伤修复和肿瘤化疗敏感性相关的lncRNA还所知甚少。近年来越来越多的研究证据表明lncRNA可以编码具有生物活性的微肽，从而调控肿瘤生长、侵袭转移。然而迄今为止，关于lncRNA编码的 微肽是否调控DNA损伤修复和肿瘤化疗耐药还未见报道。
发明公开
本发明一个目的是提供CTD-2256P15.2抑制剂的用途。
本发明提供了具有如下a-d中任一功能的抑制剂在如下1)-9)至少一种中的应用：
1)制备肿瘤治疗产品；
2)制备降低肿瘤细胞对化疗药物的耐药性的产品；
3)制备提高肿瘤细胞对化疗药物的敏感性的产品；
4)制备抑制肿瘤细胞中的DNA同源重组修复通路的产品；
5)制备抑制肿瘤细胞中的微同源介导的末端连接修复通路的产品；
6)制备抑制DNA损伤诱导的多聚ADP核糖链生成的产品；
7)制备抑制由DNA同源重组修复通路引发的肿瘤耐药性的产品；
8)制备抑制由肿瘤细胞中的微同源介导的末端连接修复通路引发的肿瘤耐药性的产品；
9)制备抑制由DNA损伤诱导的多聚ADP核糖链生成引发的肿瘤耐药性的产品；
a、抑制CTD-2256P15.2基因的表达；
b、抑制由CTD-2256P15.2基因编码的微肽PACMP的生物学功能；
c、抑制含有PACMP的融合蛋白的生物学功能；
d、抑制含有PACMP的复合物的生物学功能；
所述CTD-2256P15.2基因的核苷酸序列为序列表中序列1或序列1的第138-272位。
所述微肽PACMP的氨基酸序列为序列表中序列8。
上述肿瘤治疗产品，为肿瘤治疗药物，其功能包括抑制肿瘤细胞的增殖或诱导肿瘤细胞死亡或抑制肿瘤的生长。
进一步的，所述肿瘤治疗药物中，CTD-2256P15.2或其编码的微肽PACMP的抑制剂作为其中的唯一有效成分或者有效成分之一。
上述应用中，所述肿瘤包括但不限于乳腺癌、卵巢癌、肺癌、肝癌、胃癌、结直肠癌、头颈癌、膀胱癌、宫颈癌、弥漫型B大细胞淋巴瘤、食管癌、胶质细胞瘤、胰腺癌、前列腺癌、黑色素瘤、胸腺瘤、子宫内膜癌。
上述应用中，所述抑制剂是指能对CTD-2256P15.2或其编码微肽PACMP具有抑制效果的分子，该抑制效果包括但不限于：抑制CTD-2256P15.2的转录或翻译，促进CTD-2256P15.2或PACMP降解，抑制PACMP功能。
所述CTD-2256P15.2或微肽PACMP的抑制剂可以为siRNA、shRNA、antisense RNA、miRNA、基因敲除或敲降的CRISPR相关质粒和病毒载体、抗体、多肽、小分子化合物。
具体地，所述抑制剂为靶向CTD-2256P15.2的siRNA或shRNA或靶向PACMP编码区的gRNA或表达上述各个RNA的载体；
所述靶向CTD-2256P15.2RNA的siRNA的核苷酸序列为序列表中序列2或序列3；
所述靶向CTD-2256P15.2RNA的shRNA的核苷酸序列为序列表中序列4或序列5；
所述靶向PACMP编码区的gRNA的核苷酸序列为序列6。
本发明提供的抑制CTD-2256P15.2的siRNA和shRNA的功能是指靶向CTD-2256P15.2的RNA序列，降低其RNA水平，包括上述序列以及所有具有类似功能的siRNA和shRNA。
上述抑制剂和其他抗肿瘤物质(即为新型抗肿瘤药物组合方案)在如下至少一种中的应用：
1)制备肿瘤治疗产品；
2)制备降低肿瘤细胞对化疗药物的耐药性的产品；
3)制备提高肿瘤细胞对化疗药物的敏感性的产品；
所述其他抗肿瘤物质为其他抗肿瘤药物或其他抗肿瘤治疗方法所需的试剂或仪器。所述其他肿瘤治疗药物或方法是指能对肿瘤细胞DNA造成损伤或诱发复制压力的肿瘤治疗药物或方法，包括但不限于：蒽环类药物、喜树碱、PARP抑制剂、ATR抑制剂、CDK4/6抑制剂、放射治疗。
进一步的，抗肿瘤药物为化疗药物。
上述新型抗肿瘤药物组合方案可以是以下形式中的任意一种：
(1)将CTD-2256P15.2或其编码的微肽PACMP的抑制剂与其他肿瘤治疗药物或治疗方法分别独立施用，施用途径可相同或不同，可在其他肿瘤治疗药物或治疗方法疗程施用前、中、后独立施加上述抑制剂。
(2)将CTD-2256P15.2或其编码的微肽PACMP的抑制剂与其他肿瘤治疗药物制成复方制剂，即在CTD-2256P15.2或其编码的微肽PACMP的抑制剂和其他肿瘤治疗药物采用相同给药途径同时给药时，将两者制成复方制剂。
本发明另一个目的是提供一种产品，其包括上述抑制剂，和，其他肿瘤治疗药物或其他抗肿瘤治疗方法所需的试剂或仪器；
所述产品具有如下至少一种功能：
1)肿瘤治疗；
2)降低肿瘤细胞对化疗药物的耐药性；
3)提高肿瘤细胞对化疗药物的敏感性。
上述CTD-2256P15.2或其编码的微肽PACMP在作为治疗肿瘤试剂的作用靶点中的应用也是本发明保护的范围。
上述CTD-2256P15.2基因作为标志物在制备评估肿瘤患者对化疗药物敏感性或预测肿瘤患者预后状态产品中的应用本发明保护的范围。
本发明还有一个目的是提供检测肿瘤组织中CTD-2256P15.2的表达量的物质的用途。
本发明提供的检测肿瘤组织中CTD-2256P15.2的表达量的物质在如下中的应用：
1)制备预测肿瘤患者化疗后预后状态的产品；
2)制备评估或辅助评估肿瘤患者对化疗药物敏感性的产品。
本发明还提供了检测肿瘤组织中CTD-2256P15.2的表达量的物质和数据处理装置的应用，为1)-2)中的任一种：
1)制备预测肿瘤患者化疗后预后状态的产品；
2)制备评估或辅助评估肿瘤患者对化疗药物敏感性的产品；
所述数据处理装置内设模块；所述模块具有如下(a1)和(a2)所示的功能：
(a1)以肿瘤患者组成的待测群体的离体肿瘤组织为标本，测定每份标本中所述CTD-2256P15.2基因的表达量，然后根据基因表达量将所述待测群体分为低表达组和高表达组；
(a2)按照如下标准确定来自于所述待测群体的待测患者的预后：
所述低表达组中的待测患者化疗后的预后状态好于或候选好于所述 高表达组中的待测患者；
或，所述低表达组中的待测患者化疗后的预后总生存期长于或候选长于所述高表达组中的待测患者；
或，所述低表达组中的待测患者化疗后的预后总生存率高于或候选高于所述高表达组中的待测患者；
或，所述低表达组中的待测患者化疗后的预后无疾病进展生存期长于或候选长于所述高表达组中的待测患者；
或，所述低表达组中的待测患者化疗后的预后无疾病进展生存率高于或候选高于所述高表达组中的待测患者；
或，所述低表达组中的待测患者对化疗药物的敏感度高于或候选高于所述高表达组中的待测患者。
本发明还提供了用于预测待测肿瘤患者化疗后预后的系统，包括检测肿瘤组织中CTD-2256P15.2的表达量的物质和上述数据处理装置。
上述检测肿瘤组织中CTD-2256P15.2的表达量的物质为特异结合或扩增所述CTD-2256P15.2中的探针或引物。
上述化疗采用的化疗药物为能造成DNA损伤的肿瘤治疗药物，包括但不限于蒽环类药物、喜树碱或PARP抑制剂。
所述肿瘤病人对化疗药物敏感性或预后评价试剂中，CTD-2256P15.2基因的表达作为其中唯一检测指标或有效检测指标之一。
本发明还提供了如下方法：
本发明提供了一种治疗或辅助治疗肿瘤的方法，包括如下步骤：单独使用上述抑制剂治疗肿瘤。
本发明提供了一种治疗或辅助治疗肿瘤的方法，包括如下步骤：上述抑制剂和其他肿瘤治疗药物配合使用，治疗肿瘤。
本发明提供了一种治疗或辅助治疗肿瘤的方法，包括如下步骤：使用上述抑制剂和其他肿瘤治疗方法配合使用，治疗肿瘤。
本发明提供了一种对肿瘤病人进行化疗药物治疗后预后状态辅助评估的方法，包括如下步骤：检测肿瘤病人的肿瘤组织中的CTD-2256P15.2的表达量；
CTD-2256P15.2的表达量高的病人其预后状态差于或候选差于 CTD-2256P15.2的表达量低的病人。
本发明提供了一种对肿瘤病人进行对化疗药物敏感性评估或辅助评估的方法，包括如下步骤：检测肿瘤病人的肿瘤组织中的CTD-2256P15.2的表达量；
CTD-2256P15.2的表达量高的病人对化疗药物敏感性低于或候选低于CTD-2256P15.2的表达量低的病人。
上述检测肿瘤组织中CTD-2256P15.2的表达量的物质具体为实施例的荧光定量PCR所用引物。
所述预后状态具体体现在无疾病进展生存期和/或总体生存期，具体为：
CTD-2256P15.2基因的表达量高的肿瘤组织对应的肿瘤病人的化疗后总体生存期小于或候选小于CTD-2256P15.2基因的表达量低的肿瘤组织对应的肿瘤病人；
或，CTD-2256P15.2基因的表达量高的肿瘤组织对应的肿瘤病人的化疗后无疾病进展生存期小于或候选小于CTD-2256P15.2基因的表达量低的肿瘤组织对应的肿瘤病人。
上述化疗药物为能造成DNA损伤的肿瘤治疗药物，包括但不限于蒽环类药物、喜树碱或PARP抑制剂；
进一步的，耐药性为耐化疗药。
CTD-2256P15.2在多种肿瘤类型中高表达，且能被多种DNA损伤化疗药物诱导表达。CTD-2256P15.2可以编码一个功能性的微肽PACMP。PACMP一方面通过与泛素连接酶Cul3的底物衔接蛋白KLHL15结合，竞争性抑制后者与同源重组修复通路中重要因子CtIP的结合和泛素化降解，另一方面通过与化疗和DNA损伤诱发的PAR链结合，促进PAR信号放大，二者协同促进肿瘤细胞的生长和耐药。因此CTD-2256P15.2或其编码的微肽PACMP是抑制肿瘤生长和增强肿瘤临床治疗效果的新靶标。
附图说明
图1为CTD-2256P15.2在表阿霉素耐药乳腺癌细胞系MCF7-EPI中高表达。
图2为CTD-2256P15.2的表达与乳腺癌病人的预后负相关。
图3为敲低CTD-2256P15.2能增强多种肿瘤细胞对表阿霉素药物的敏感性。
图4为敲低CTD-2256P15.2能增加肿瘤细胞对多种治疗方案(包括化疗、靶向治疗和放疗)的敏感性。
图5为CTD-2256P15.2能编码微肽。
图6为CTD-2256P15.2通过其编码的微肽调控肿瘤细胞的化疗敏感性。
图7为抑制CTD-2256P15.2或其编码的微肽能显著抑制肿瘤生长和增强化疗药物敏感性。
图8为抑制CTD-2256P15.2或其编码的微肽能降低同源重组修复和微同源末端连接修复效率、降低CtIP蛋白水平和DNA损伤诱导的PAR水平。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的总生存期(Overall Survival，OS)定义为从入组至任何原因导致的死亡或末次随访时间。
下述实施例中的总生存率定义为患者从某一特定时点开始随访，到某一特定时间尚能生存的概率。
下述实施例中的无疾病进展生存期(PFS)定义为肿瘤疾病患者从接受治疗开始，到观察到疾病进展或者发生因为任何原因的死亡之间的这段时间。
下述实施例中的无疾病进展生存率定义为患者从某一特定时点开始随访，到某一特定时间观察不到疾病进展的概率。
实施例1、长链非编码RNA CTD-2256P15.2及其作为化疗预后状态预测的标志物
一、长链非编码RNA CTD-2256P15.2的发现
通过对蒽环类化疗药物表阿霉素敏感和耐药的乳腺癌肿瘤组织进行转录组测序，鉴定到长链非编码RNA CTD-2256P15.2在表阿霉素耐药的肿瘤组织中高表达。
长链非编码RNA CTD-2256P15.2的核苷酸序列为序列表中序列1。
二、验证CTD-2256P15.2表达与肿瘤耐药性的关系
利用CCK8方法检测肿瘤细胞对表阿霉素的耐药性，具体如下：
将处于对数期的MCF7-EPI细胞(通过在MCF7细胞培养过程中逐步提高表阿霉素浓度(从20nM到500nM)得到的耐表阿霉素的细胞)和对照细胞MCF7(ATCC Cat#HTB-22,RRID:CVCL_0031)分别接种到96孔板中，每孔4000个细胞。培养12h后，分别向每个培养孔的培养体系中加入不同浓度表阿霉素(EPI)。继续培养24小时后，吸净培养基，加入含有10％(体积百分含量)CCK8试剂(DOJINDO，Cat#CK04)的新鲜培养基，孵育4小时。然后用酶标仪检测每孔在465nm的吸光值，绘制细胞相对存活曲线。
结果如图1A所示，可以看出，随着EPI浓度增加，表阿霉素耐药的细胞系MCF7-EPI的存活率高于细胞系MCF7，也就是证明MCF7-EPI对EPI耐药。
用Trizol试剂提取MCF7-EPI细胞和MCF7细胞系总RNA，用反转录酶将提取的RNA反转录成cDNA,然后用实时荧光定量PCR检测CTD-2256P15.2的RNA水平。
实时荧光定量PCR检测的引物为CTD-2256P15.2：正向引物：GACTTCTGCATTTGGCTGGAAGG,反向引物：CTAACTCAGGGTATCGGAACCGA；内参GAPDH：正向引物：GGAGCGAGATCCCTCCAAAAT，反向引物：GGCTGTTGTCATACTTCTCATGG。
结果如图1B所示，可以看出，CTD-2256P15.2在MCF7-EPI中的表达显著高于对照MCF7细胞。
三、CTD-2256P15.2的表达与肿瘤病人化疗敏感性的关系
为了进一步验证CTD-2256P15.2的表达与肿瘤病人化疗敏感性的关系，收集了92例接受过以表阿霉素为基础的化疗方案的乳腺癌病人肿瘤组织(来源于天津医科大学肿瘤医院)，其中48例病人在化疗后肿瘤无进展，预后较好，为预后好组；44例病人化疗后疾病发生进展，预后较差，为预后差组(表1和表2)。
表1为44例预后差组乳腺癌病人肿瘤组织的表达量的结果
Figure PCTCN2021130604-appb-000001
Figure PCTCN2021130604-appb-000002
Figure PCTCN2021130604-appb-000003
上表中，第3列中复发状态的1表示第2列随访时间内复发，0表示第2列随访时间内无复发或者失访；第5列中死亡状态的1表示第4列随访时间内死亡，0表示第4列随访时间内未死亡或者失访。
表2为48例预后好组乳腺癌病人肿瘤组织的表达量的结果
Figure PCTCN2021130604-appb-000004
Figure PCTCN2021130604-appb-000005
上表中，第3列中复发状态的1表示第2列随访时间内复发，0表示 第2列随访时间内无复发或者失访；第5列中死亡状态的1表示第4列随访时间内死亡，0表示第4列随访时间内未死亡或者失访。
上述92例乳腺癌肿瘤组织样品为福尔马林固定石蜡包埋的组织切片，用NucleoSpintotal RNA extraction试剂盒提取肿瘤组织RNA，具体提取步骤参照试剂盒的使用说明。接着用反转录酶将提取的RNA反转录成cDNA，然后用实时荧光定量PCR检测CTD-2256P15.2在这些肿瘤组织中的表达情况。
荧光定量PCR技术的方法同上述二实时荧光定量PCR。
将表达量高于所有患者表达量中位值(中位值为1.15)的患者归为高表达病人组，表达量低于等于所有患者表达量中位值的患者归为低表达病人组。
结果如图2所示，A为CTD-2256P15.2在预后差和预后好的病人中的表达量，B为无疾病进展生存期生存期(PFS),C为总体生存期(OS)，可以看出，CTD-2256P15.2在预后差的病人肿瘤组织中表达显著高于预后好的病人组织(图2A)；通过分析这些肿瘤病人的预后情况，发现与CTD-2256P15.2低表达的病人相比，肿瘤组织中CTD-2256P15.2高表达的病人的无疾病进展生存期(PFS,图2B，反应在无疾病进展生存率)和总体生存期(OS,图2C，反应在总体生存率)更短，预后较差，反应了CTD-2256P15.2表达量高的病人对药物敏感性差，导致疾病进展。
因此，检测肿瘤组织CTD-2256P15.2基因的表达量可以预测肿瘤病人对化疗药物的敏感性高低；或检测肿瘤组织CTD-2256P15.2基因的表达量可以预测肿瘤病人的预后状态，具体体现在无疾病进展生存期和/或总体生存期；
CTD-2256P15.2基因的表达量高的肿瘤组织对应的肿瘤病人对化疗药物的敏感性小于CTD-2256P15.2基因的表达量低的肿瘤组织对应的肿瘤病人；
CTD-2256P15.2基因的表达量高的肿瘤组织对应的肿瘤病人化疗药物治疗后的预后状态差于CTD-2256P15.2基因的表达量低的肿瘤组织对应的肿瘤病人；
具体为CTD-2256P15.2基因的表达量高的肿瘤组织对应的肿瘤病人的 化疗后总体生存期小于CTD-2256P15.2基因的表达量低的肿瘤组织对应的肿瘤病人；
或，CTD-2256P15.2基因的表达量高的肿瘤组织对应的肿瘤病人的化疗后无疾病进展生存期小于CTD-2256P15.2基因的表达量低的肿瘤组织对应的肿瘤病人。
实施例2、CTD-2256P15.2作为治疗肿瘤的靶点
一、CTD-2256P15.2在多种肿瘤中的表达情况
为了验证抑制CTD-2256P15.2能增加多种肿瘤对化疗药物的敏感性，首先分析了CTD-2256P15.2在多种肿瘤中的表达情况。
在GePIA网站检索TCGA多种肿瘤的转录组测序定量数据。CTD-2256P15.2基因的Ensembl ID为ENSG00000259802，通过检索该ID可以得到CTD-2256P15.2在多种肿瘤类型组织中的表达情况。BLCA：膀胱尿路上皮癌；BRCA：乳腺浸润癌；CESC：宫颈鳞癌和腺癌；COAD：结肠癌；DLBC：弥漫性大B细胞淋巴癌；ESCA:食管癌；GBM:多形成性胶质细胞瘤；HNSC:头颈鳞状细胞癌；LGG:脑低级别胶质瘤；LUAD：肺腺癌；LUSC:肺鳞癌；OV:卵巢浆液性囊腺癌；PAAD:胰腺癌；PRAD:前列腺癌；READ:直肠腺癌；STAD:胃癌；THYM:胸腺癌；UCEC:子宫内膜癌；UCS:子宫肉瘤。
结果如图3A所示，TPM:Transcripts Per Million；可以看出，在乳腺癌、肺癌、胃癌、卵巢癌等多个肿瘤类型的肿瘤组织中CTD-2256P15.2基因的表达高于正常组织。
因此，多个肿瘤中可选择CTD-2256P15.2作为治疗靶点进行如下实验。
二、CTD-2256P15.2抑制剂siRNA在提高肿瘤细胞对化疗敏感性中的应用
1、CTD-2256P15.2抑制剂siRNA的制备
为了检测CTD-2256P15.2对肿瘤细胞化疗敏感性的调控作用，根据CTD-2256P15.2的RNA序列设计并合成了两条靶向CTD-2256P15.2的特异性siRNA(CTD-2256P15.2抑制剂siRNA)silnc15.2-1和silnc15.2-2,序列为：
silnc15.2-2,GCGGCUUCUGGAGGGACAA(序列2)；
silnc15.2-1,GCAGAUGACCUAGCACAAA(序列3)；
对照siRNA(siNC)的序列为：UUCUCCGAACGUGUCACGU。
2、转染siRNA且化疗敏感性实验
利用lipofectmine RNAiMAX(Invitrogen，Cat#13778150)分别将对照siRNA(siNC)、靶向CTD-2256P15.2的特异性silnc15.2-1和silnc15.2-2转染进乳腺癌细胞(MCF7 ATCC Cat#HTB-22,RRID:CVCL_0031、MDA-MB-231 ATCC Cat#CRM-HTB-26,RRID:CVCL_0062)、骨肉瘤细胞(U2OS Cat#HTB-96；RRID:CVCL_0042)、胃癌(AGS ATCC Cat#CRL-1739,RRID:CVCL_0139)、宫颈癌细胞(HeLa Cat#CRL-7923；RRID:CVCL_0030)、卵巢癌(SK-OV-3Cat#HTB-77；RRID:CVCL_0532)和非小细胞肺癌细胞(A549Cat#CCL-185；RRID:CVCL_0023)中，使siRNA工作浓度为100nM；具体为在一个6cm培养皿(约10^6细胞)使用3ml转染体系，其中siRNA工作浓度100nM来进行敲低。
在不同siRNA转染48小时后，部分细胞胰酶消化，离心收集细胞，提取细胞总RNA并反转录成cDNA，荧光定量PCR检测转染后细胞中CTD-2256P15.2水平。
荧光定量PCR检测方法实施例1，不同siRNA转染MCF7细胞的结果如图3B所示，可以看出，silnc15.2-1和silnc15.2-2实现肿瘤细胞中CTD-2256P15.2的敲低，降低其表达量。
同时在转染24小时后，将部分细胞接种到96孔板中，待细胞贴壁后向细胞培养体系中加入不同浓度表阿霉素持续处理24小时，利用CCK8方法检测细胞的存活情况(方法同实施例1的二)。
CCK8方法检测不同肿瘤细胞转染CTD-2256P15.2抑制剂siRNA的存活情况结果如图3C-3I所示，可以看出，与对照siRNA相比，CTD-2256P15.2抑制性siRNA敲低不同肿瘤细胞中CTD-2256P155.2均能显著增强这些细胞的表阿霉素敏感性，降低肿瘤细胞存活率；表明CTD-2256P15.2是增强肿瘤化疗药物杀伤作用，改善临床治疗效果的有效靶点。
因此，敲除或抑制CTD-2256P15.2表达的siRNA可以增强肿瘤细胞对化疗药物的敏感性，降低化疗药物处理后肿瘤细胞存活率。
三、CTD-2256P15.2抑制性siRNA和其他治疗配合使用
按照上述二的方法利用lipofectmine RNAiMAX(Invitrogen，Cat#13778150)分别将合成的对照siRNA(siNC)、靶向CTD-2256P15.2的特异性silnc15.2-1和silnc15.2-2转染进MCF7细胞中，使siRNA工作浓度为100nM。
转染24小时后将转染后细胞接种到96孔板中，待细胞贴壁后，分别向部分细胞的培养体系中加入不同浓度的喜树碱(Camptothecin,CPT)(Sigma,Cat#C9911)、ATR抑制剂(VE-822)(Selleck,Cat#S7102)或CDK4/6抑制剂(Palbociclib，Selleck,Cat#S1116)，持续处理48小时后，利用CCK8试剂的方法检测细胞存活情况，检测方法同实施例1。
结果如图4A-图4C所示，A,B,C分别为siRNA+喜树碱(CPT)、siRNA+ATR抑制剂(VE-822)、siRNA+CDK4/6抑制剂(Palbociclib)，可以看出，CTD-2256P15.2抑制性siRNA抑制CTD-2256P15.2的表达后能显著增加MCF7细胞对喜树碱(CPT)、ATR抑制剂(VE-822)和CDK4/6抑制剂(Palbociclib)这些肿瘤治疗药物的敏感性。
因此，这些结果表明，CTD-2256P15.2抑制性siRNA与其他放化疗方案或化疗药物组合作用能显著增强对肿瘤细胞的杀伤作用，为提升肿瘤治疗效果提供了全新策略。也就是说，CTD-2256P15.2抑制性siRNA能够增强抗肿瘤药物对肿瘤细胞的杀伤作用，降低肿瘤细胞的化疗耐药性。
四、CTD-2256P15.2抑制剂shRNA在提高肿瘤细胞对化疗敏感性中的应用
利用shRNA技术降低CTD-2256P15.2的表达水平,通过克隆形成实验检测Olaparib或X射线处理后肿瘤细胞的存活情况。具体步骤如下：
1、设计并合成两条用于靶向CTD-2256P15.2的shRNA编码序列，其中shlnc15.2-2：GCGGCTTCTGGAGGGACAAAGCTCGAGCTTTGTCCCTCCAGAAGCCGC(序列4)；shlnc15.2-1：GCAGATGACCTAGCACAAATACTCGAGTATTTGTGCTAGGTCATCTGC(序列5)。
shNC序列：TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA
将其分别连接到pLKO载体(Addgene Cat#10878https://www.addgene.org/protocols/plko/)上，获得重组的pLKO质粒，用于表达shRNA。
重组的pLKO-shlnc15.2-2质粒为将序列4所示的shlnc15.2-2的编码基因替换pLKO载体的AgeI和EcoRI位点间得到的载体，该载体表达shlnc15.2-2(序列4编码的RNA)。
重组的pLKO-shlnc15.2-1质粒为将序列5所示的shlnc15.2-1的编码基因替换pLKO载体的AgeI和EcoRI位点间得到的载体，该载体表达shlnc15.2-1(序列5编码的RNA)。
重组的pLKO-shNC质粒为将shNC的编码基因替换pLKO载体的AgeI和EcoRI位点间得到的载体。
2、慢病毒包装与感染
转染前一天在10cm培养皿中接种对数生长期的293T细胞(Cat#CRL-3216；RRID:CVCL_0063)，使细胞密度为50％左右，培养过夜后进行质粒转染。
转染前将培养基更换为不含抗生素的新鲜培养基，置于37度。按照vigoFect转染试剂说明书将5μg质粒(上述pLKO-shlnc15.2-1或pLKO-shlnc15.2-2与病毒包装质粒pRSV-Rev(Addgene ID 12253)、pMD2.G(Addgene ID 12259)、pMDLg/pRRE(Addgene ID 12251)(比例为2:1:1:1)转进293T细胞。转染24小时后，弃掉培养基，加入8ml新鲜的完全培养基继续培养。48小时后，细胞培养上清中含有相应慢病毒。
提前一天将MCF7细胞接种到6cm培养皿中，使其密度为50％，用于感染慢病毒。第二天感染时，收集293T培养上清，经0.45μM滤器过滤至干净的离心管中。将过滤后的病毒与等体积新鲜培养基混合，并加入终浓度为10μg/mL的polybrene，混匀后加入MCF7细胞中，感染后8小时换液。继续培养48小时后，加入1mg/ml的puromycin进行筛选，当细胞可以稳定在puromycin中生长且qPCR可以检测到很好的敲低效果，此时视为获得稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1/2的MCF7细胞，命名为MCF7 shRNA-1和MCF7 shRNA-2。
以转入重组的pLKO-shNC质粒的细胞为对照，命名为MCF7 shNC。上述qPCR检测方法如下：提取上述细胞的总RNA，利用反转录酶将RNA反转录成cDNA,荧光定量PCR检测CTD-2256P15.2的含量(引物同前)，结果见图4D，可以看出，shlnc15.2-1和shlnc15.2-2可以实现敲除 CTD-2256P15.2，降低其表达。
3、功能
将上述2得到的MCF7 shRNA-1或MCF7 shRNA-2以及MCF7 shNC(对照细胞)分别接种到6cm培养皿中。
细胞贴壁后，部分约500个细胞加入不同浓度的PARP抑制剂奥拉帕尼(Olaparib)处理24小时，弃掉培养基，用PBS洗细胞2次，加入新鲜的完全培养基，置于37度培养箱中继续培养14天。弃掉培养基，用水轻轻润洗细胞2次，室温晾干培养皿，对肉眼可见的细胞克隆个数进行统计，并按照公式(存活比例＝不同处理条件下形成的克隆数除以同组不处理条件下形成的克隆数)计算克隆存活比例，绘制克隆存活曲线。
或将上述部分约500个贴壁的细胞照射不同剂量的X射线造成DNA损伤，继续培养14天，统计细胞克隆个数，绘制克隆存活曲线。
结果如图4E和4F所示，利用shRNA降低MCF7细胞中CTD-2256P15.2的表达能显著增强PARP抑制剂和X射线对细胞的杀伤作用，降低肿瘤细胞存活，增加细胞对PARP抑制剂化疗和射线放疗的敏感性。
上述结果表明，CTD-2256P15.2抑制剂shRNA与其他放化疗方案或化疗药物组合作用能显著增强对肿瘤细胞的杀伤作用，为提升肿瘤治疗效果提供了全新策略。也就是说，CTD-2256P15.2抑制剂shRNA能够强抗肿瘤药物或肿瘤治疗方法对肿瘤细胞的杀伤作用，降低肿瘤细胞的化疗耐药性。
实施例3、CTD-2256P15.2编码的微肽及其调控肿瘤细胞的化疗敏感性
一、CTD-2256P15.2编码的微肽
通过生物信息预测发现CTD-2256P15.2的开放式阅读框1(ORF1，即序列1第138-272位)可能编码一个44氨基酸长度的微肽，该微肽的氨基酸序列为MAASGGTKKA QSGGRRLREPSSRPSRRARQRPRRGALRKAGRFL(序列8)。
为了验证CTD-2256P15.2的编码性，利用CRISPR-Cas9技术在U2OS细胞的CTD-2256P15.2基因ORF1的3’端末尾(终止密码子前)敲入一段含SBP-FLAG标签的序列。通过挑取单克隆细胞并进行PCR测序鉴定，获得了插入成功的单克隆细胞。
具体方法如下：
1)设计并合成一段特异性靶向ORF1 3’端末尾的DNA序列： CGGCGTGCACTGTCGGTCGGCGG(序列6)，将此DNA克隆到pX330载体上，用以转录生成gRNA。
重组载体pX330-gRNA为将CGGCGTGCACTGTCGGTCGGCGG(序列6)所示的DNA分子插入pX330载体(Addgene ID 42230)的Bbs1位点间，得到的载体，其表达gRNA(序列6编码的gRNA)。
2)设计并合成donor DNA：
donor DNA为左同源臂-含标签的序列(SBP-FLAG-P2A-puromycin)-右同源臂，核苷酸序列为序列7。
3)敲入
将上述donor DNA和pX330-gRNA质粒采用Pulse Generator-CUY21EDDID 2电转仪器，电压135V通过电穿孔的方式转进U2OS细胞(ATCC，Cat#HTB-96；RRID:CVCL_0042)中。48小时后，加入1mg/ml的puromycin进行筛选，然后将puromycin筛选后生长的细胞进行单克隆接种。2周后，挑取单克隆细胞并扩大培养，然后提取基因组DNA，利用PCR扩增和测序鉴定敲入成功的阳性克隆U2OS-KI细胞。
上述PCR扩增采用的引物为：正向引物：AGAGGCTGACAGAAAGCGAG，反向引物：CTAACTCAGGGTATCGGAACCGA，得到大约1580bp片段并且测序含有敲入标签序列的为敲入成功的阳性克隆，命名为U2OS-KI细胞，该细胞表达融合蛋白ORF1-SBP-FLAG-P2A(该融合蛋白的编码核酸为序列10，其为将CTD-2256P15.2第一开放阅读框的3'末端(终止密码子之前)融合SBP-FLAG-P2A-puromycin得到的序列，在蛋白翻译时，P2A序列发生自剪切断裂，从而获得ORF1-SBP-FLAG-P2A融合蛋白)。
4)shCTD-2256P15.2细胞
shRNA技术敲低上述3)得到的U2OS-KI细胞中的CTD-2256P15.2，方法参考实施例2的四，采用重组的pLKO-shlnc15.2-1质粒通过慢病毒转染U2OS-KI细胞后得到的细胞，命名为U2OS-KI-shlnc15.2。
采用重组的pLKO-shNC质粒通过慢病毒转染U2OS-KI细胞后得到的细胞，命名为U2OS-KI-shNC。
5)检测表达
A、U2OS-KI对照细胞中验证ORF1的内源表达。具体方法如下：
(1)免疫荧光：将对数期的U2OS细胞(对照细胞)、U2OS-KI-shlnc15.2和U2OS-KI-shNC细胞60-80％密度接种到玻片上。第二天，吸净培养基，PBS洗细胞两次，加入4％多聚甲醛溶液室温固定细胞20分钟。弃去固定液，PBS洗细胞3次。加入含0.5％的Triton X-100的PBS溶液，室温处理细胞10分钟，弃去Triton溶液，PBS洗细胞3次，加入含5％BSA的PBS溶液室温封闭处理1小时。然后按顺序先后将细胞与一抗SBP标签抗体(Santa Cruz,Cat#sc-101595；RRID:AB_1128239)、荧光二抗(Life Technologies,Cat#A11029；RRID:AB_138404)进行孵育，最后用含DAPI的封闭剂封片。
显微镜检测ORF1融合蛋白的表达情况。
结果如图5A所示，WT为U2OS细胞，KI-shlnc15.2为U2OS-KI-shlnc15.2细胞，KI-shNC为U2OS-KI-shNC细胞，可以看出，在U2OS-KI-shNC细胞中，ORF1能够起始翻译，表达ORF1-SBP-FLAG-P2A融合蛋白，而lnc15.2敲低的细胞U2OS-KI-shlnc15.2中没有融合蛋白的表达。
(2)免疫沉淀：收集4ⅹ10 6对数生长期的U2OS、U2OS-KI-shNC、U2OS-KI-shlnc15.2细胞，用含1％Triton的裂解缓冲液裂解细胞并离心，向获得的细胞裂解液中加入anti-FLAG beads，4度旋转孵育2小时。离心弃去上清，用裂解缓冲液清洗beads后，加入2×SDS样品缓冲液，95度煮10分钟。用western blot技术检测ORF1融合蛋白的表达情况。
结果如图5B所示，在U2OS-KI-shNC中，ORF1能够起始翻译，表达ORF1-SBP-FLAG-P2A融合蛋白,sh15.2敲低的细胞U2OS-KI-shlnc15.2中ORF1-SBP-FLAG-P2A的表达相比于U2OS-KI-shNC细胞显著降低。
这些结果表明，CTD-2256P15.2的ORF1能编码一个44氨基酸长度的微肽。
二、CTD-2256P15.2是通过其编码的微肽来调控肿瘤细胞的化疗敏感性
为了验证CTD-2256P15.2编码的微肽对肿瘤细胞化疗敏感性的调控作用，在CTD-2256P15.2稳定敲低的MCF7和U2OS细胞中回补表达能抵抗shCTD-2256P15.2的CTD-2256P15.2野生型全长(FL)和ORF1起始密码子 突变的全长(FL*)，以及回补ORF1融合蛋白，检测细胞对表阿霉素的敏感性。
具体方法如下：
1、构建CTD-2256P15.2稳定敲低的MCF7和U2OS细胞
利用shRNA技术构建CTD-2256P15.2稳定敲低的MCF7和U2OS细胞，具体步骤参照实例2。不同的是细胞采用MCF7、U2OS细胞，重组质粒为pLKO-shlnc15.2-1质粒，得到稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MCF7细胞和稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的U2OS细胞。
以重组的pLKO-shNC质粒敲除为对照，得到对照表达shNC的MCF7细胞和对照表达shNC的U2OS细胞。
2、构建回补细胞系
具体步骤如下：
1)将抵抗shRNA的CTD-2256P15.2野生型全长(FL)、ORF1起始密码子突变(ATG突变成ATT，不能再起始编码微肽)的全长(FL*)以及ORF1融合蛋白(ORF1C端融合SBP-FLAG标签)分别连接到pNL慢病毒表达载体(pNL-EGFP/CMV/WPREdU3,Addgene,#17579)上，筛选获得重组质粒，具体如下：
重组质粒FL为将CTD-2256P15.2的基因序列(序列1)克隆到pNL慢病毒表达载体的Nhe1和EcoR1酶切位点间得到的载体；
重组质粒FL*为将CTD-2256P15.2的ORF1起始密码子突变序列(其序列为序列1的第138-141位所示序列的ATG突变为ATT，即1号开放阅读框架的起始密码子ATG突变为ATT)，然后克隆到pNL慢病毒表达载体的Nhe1和EcoR1酶切位点间得到的载体；
重组质粒ORF1为将ORF1-SBP-FLAG融合蛋白的编码基因(其序列为序列9，即序列1第138-269位+SBP-FLAG核酸序列)克隆到pNL慢病毒表达载体的Nhe1和EcoR1酶切位点间得到的载体；
2)将上述各个重组质粒通过慢病毒包装与感染的方法(方法同实施例2的四的2)分别与包装质粒共转染到293T细胞中，获得含有病毒颗粒的培养上清；再将慢病毒分别感染稳定表达靶向CTD-2256P15.2的 shRNA(shCTD-2256P15.2)-1的MCF7细胞和稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的U2OS细胞，获得稳定回补细胞，分别命名为ORF1回补shRNA(shCTD-2256P15.2)-1的MCF7细胞(转入重组质粒ORF1)、ORF1回补shRNA(shCTD-2256P15.2)-1的U2OS细胞(转入重组质粒ORF1)、Fl*回补shRNA(shCTD-2256P15.2)-1的MCF7细胞(转入重组质粒FL*)、Fl*回补shRNA(shCTD-2256P15.2)-1的U2OS细胞(转入重组质粒FL*)、Fl回补shRNA(shCTD-2256P15.2)-1的MCF7细胞(转入重组质粒FL)和Fl回补shRNA(shCTD-2256P15.2)-1的U2OS细胞(转入重组质粒FL)。
将上述细胞接种到96孔板中，用1μM表阿霉素处理贴壁后的细胞24小时，利用CCK8试剂检测细胞的存活率，方法同实施例1。以对照表达shNC的MCF7细胞、对照表达shNC的U2OS细胞、稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MCF7细胞和稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的U2OS细胞作为对照。
结果如图6所示，A为MCF7细胞，B为U2OS细胞；shNC表示表达shNC的细胞，shlnc15.2表示稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的细胞，shlnc15.2+ORF1表示ORF1回补shRNA(shCTD-2256P15.2)-1的细胞，shlnc15.2+FL表示FL回补shRNA(shCTD-2256P15.2)-1的细胞，shlnc15.2+FL*表示FL*回补shRNA(shCTD-2256P15.2)-1的细胞，可以看出，敲低CTD-2256P15.2能增加肿瘤细胞对表阿霉素的敏感性，回补表达野生型全长(ORF1)和ORF1融合蛋白(FL)能回复降低细胞的表阿霉素敏感性，然而回补表达ORF1突变(FL*)的全长不能回复。
上述结果表明，CTD-2256P15.2是通过其编码的微肽来调控肿瘤细胞的化疗敏感性。
三、动物水平验证CTD-2256P15.2或其编码的微肽对肿瘤化疗敏感性的调控作用
构建了CTD-2256P15.2稳定敲低以及野生型全长或ORF1突变型全长回补的三阴性乳腺癌细胞系MDA-MB-231，并将一定量细胞接种到雌裸鼠的乳腺脂肪垫下。待移植瘤长到一定大小，分别用生理盐水和表阿霉素处理 小鼠，测量并统计肿瘤的大小。在实验终点，处死小鼠并解剖出肿瘤，进行拍照并测量肿瘤重量。
具体如下：
1)利用shRNA构建CTD-2256P15.2稳定敲低的MDA-MB-231细胞，以及在敲低细胞中通过慢病毒感染构建CTD-2256P15.2野生型全长(FL)回补或ORF1起始密码子突变(从ATG突变为ATT)的全长突变型(FL*)回补的MDA-MB-231细胞，具体如下：
参照实施例2，将重组的pLKO-shlnc15.2-1质粒转入MDA-MB-231细胞系，得到稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MDA-MB-231细胞；
以重组的pLKO-shNC质粒转染为对照，得到表达shNC的对照敲低MDA-MB-231细胞。
参考上述二的2，将野生型lnc15.2全长(FL)和突变型lnc15.2全长(FL*)分别转入稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MDA-MB-231细胞，得到FL回补shRNA(shCTD-2256P15.2)-1的MDA-MB-231细胞和FL*回补shRNA(shCTD-2256P15.2)-1的MDA-MB-231细胞。
2)将4×10 6对数生长期的上述细胞，与等体积基质胶混合，接种到雌性BALB/c裸鼠的乳腺脂肪垫下。待肿瘤的长径达到5mm，将小鼠随机分成两组，分别腹腔注射5mg/kg表阿霉素或等体积生理盐水。每4-5天给药一次，每隔一天测量肿瘤体积，绘制肿瘤相对生长曲线。在初次给药后第11天处死小鼠，解剖出肿瘤，拍照并测量肿瘤重量。以表达shNC的MDA-MB-231细胞和稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MDA-MB-231细胞为对照。
结果如图7所示，A-C分别为(A)移植的各组肿瘤细胞在表阿霉素处理或不处理情况下肿瘤组织的生长情况，(B)收集的各组移植瘤的重量，(C)收集的各组移植瘤的拍照。shNC表示表达shNC的MDA-MB-231细胞，shlnc15.2表示稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MDA-MB-231细胞，shlnc15.2+FL表示在shlnc5.2MDA-MB-231细胞中回补野生型全长lnc15.2，shlnc15.2+FL*表 示在shlnc5.2MDA-MB-231细胞中回补突变型全长lnc15.2，无论从肿瘤体积、肿瘤还是肿瘤大小，可以看出，敲低CTD-2256P15.2显著抑制肿瘤的生长和增加对表阿霉素的敏感性，回补野生型而不是突变型全长可以回复肿瘤的生长和对表阿霉素的敏感性。
这表明抑制CTD-2256P15.2或其编码的微肽有优秀的临床应用潜能，单独抑制即可降低肿瘤生长，而与其他化疗药物联合应用能增强化疗药物的杀伤作用，改善临床治疗效果。
四、研究了CTD-2256P15.2或其编码的微肽调控肿瘤生长和对化疗药物敏感性的作用机制
1)DNA同源重组修复(HR)效率和微同源介导的末端链接(MMEJ)检测
在DR-U2OS细胞(记载在如下文献中：Xia,B.,Sheng,Q.,Nakanishi,K.,Ohashi,A.,Wu,J.,Christ,N.,Liu,X.,Jasin,M.,Couch,F.J.,andLivingston，D.M.(2006).Control of BRCA2 cellular and clinical functions by a nuclear partner,PALB2.Mol Cell 22,719-729.)和U2OS EGFP-MMEJ细胞(记载在如下文献中：Wang，H.,Shao,Z.,Shi,L.Z.,Hwang,P.Y.,Truong,L.N.,Berns,M.W.,Chen,D.J.C.,and Wu,X.(2012).CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks.J Biol Chem.287(25),21471-21480.)中，分别将silnc15.2-1、silnc15.2-2和siRNA(siNC)转染进细胞(方法同实施例2的二)，得到silnc15.2-1敲低DR-U2OS细胞、silnc15.2-2敲低DR-U2OS细胞、siNC敲除DR-U2OS细胞、silnc15.2-1敲低MMEJ细胞、silnc15.2-2敲低MMEJ细胞和siNC敲除MMEJ细胞。
以导入siBRCA1(UAUAAGACCUCUGGCAUGAAU)和siCtIP(GCUAAAACAGGAACGAAUC)为阳性对照。
用流式细胞仪分选GFP阳性的细胞，并分别对GFP阳性细胞以及总细胞进行计数，阳性细胞占总细胞的比例即为DNA同源重组修复(HR)效率或微同源介导的末端链接(MMEJ)效率。
结果如图8A(DR-U2OS细胞)和8B(U2OS EGFP-MMEJ细胞)所示，利用siRNA敲低CTD-2256P15.2后，DNA同源重组修复(HR)效率和微同 源介导的末端链接(MMEJ)效率显著降低。
2)HR修复关键因子CtIP的蛋白含量检测
上述二制备的稳定表达靶向CTD-2256P15.2的shRNA(shCTD-2256P15.2)-1的MCF7细胞、ORF1回补shRNA(shCTD-2256P15.2)-1的MCF7细胞(转入重组质粒ORF1)、Fl*回补shRNA(shCTD-2256P15.2)-1的MCF7细胞(转入重组质粒FL*)，收集对数生长期细胞，通过western blot方法检测细胞内CtIP的蛋白水平。
结果如图8C所示，利用shRNA抑制CTD-2256P15.2表达后，MCF7细胞中的HR修复关键因子CtIP的蛋白含量显著降低，回补表达野生型全长而不是ORF1突变型全长可以回复CtIP的蛋白水平。
3)PAR检测
将pNL慢病毒空表达载体(即为pNL慢病毒表达载体)、重组质粒FL和重组质粒FL*分别转入silnc15.2-1敲低MCF7细胞，得到pNL慢病毒空表达载体回补silnc15.2-1的MCF7细胞、FL回补silnc15.2-1的MCF7细胞和FL*回补silnc15.2-1的MCF7细胞。
将上述对数生长期的细胞接种到6孔板中，使细胞密度为70％左右。第二天，弃掉培养基，用含400μM H 2O 2的无血清培养基于37度处理细胞5分钟，弃掉培养基，PBS洗细胞两次，加入1×SDS样品缓冲液裂解细胞。通过western blot检测细胞内PAR的水平。以不含H 2O 2的无血清培养基作对照。
结果如图8D所示，siNC为siNC处理的MCF7细胞，silnc15.2为silnc15.2-1处理的MCF7细胞、-为pNL慢病毒空表达载体回补silnc15.2-1的MCF7细胞，FL为FL回补silnc15.2-1的MCF7细胞、FL*为FL*回补silnc15.2-1的MCF7细胞；可以看出敲低CTD-2256P15.2后能显著抑制DNA损伤诱导的多聚ADP核糖(PAR)修饰链的生成，野生型全长可以回复PAR生成，ORF1突变型全长不能回复PAR(图8D)。
上述结果表明，CtIP是细胞正常起始DNA同源重组修复和微同源介导的末端链接修复功能的关键因子，CtIP缺失后两种DNA损伤修复通路受损。抑制CTD-2256P15.2或其编码的微肽PACMP导致的CtIP和/或PAR的同时下调能通过合成致死的效应达到抑制肿瘤生长的效果。
工业应用
本发明提供的CTD-2256P15.2或其编码的微肽PACMP的抑制剂在作用于肿瘤细胞或肿瘤组织时，能显著抑制肿瘤细胞的生长，增加肿瘤细胞的凋亡，缩小肿瘤体积，具有优异的抗肿瘤效果。本发明提供的新型抗肿瘤药物组合方案，将CTD-2256P15.2或其编码微肽PACMP的抑制剂和其他抗肿瘤药物联合使用，能显著增强抗肿瘤药物对肿瘤细胞的杀伤作用，降低肿瘤细胞的化疗耐药性，从而改善肿瘤临床治疗效果。CTD-2256P15.2在化疗耐药的肿瘤组织和细胞系中高表达，且其高表达与肿瘤病人的无疾病进展生存期和总体生存期显著负相关。本发明提供的CTD2256P15.2基因表达水平可作为预测肿瘤病人对化疗敏感性及预后的分子指标的应用，为有效指导肿瘤病人的临床化疗用药，评价治疗预后开创了新标准。

Claims (15)

  1. 具有如下a-d中任一功能的抑制剂在如下1)-9)至少一种中的应用：
    1)制备肿瘤治疗产品；
    2)制备降低肿瘤细胞对化疗药物的耐药性的产品；
    3)制备提高肿瘤细胞对化疗药物的敏感性的产品；
    4)抑制肿瘤细胞中的DNA同源重组修复通路；
    5)抑制肿瘤细胞中的微同源介导的末端连接修复通路；
    6)抑制DNA损伤诱导的多聚ADP核糖链生成；
    7)制备抑制由DNA同源重组修复通路引发的肿瘤耐药性的产品；
    8)制备抑制由肿瘤细胞中的微同源介导的末端连接修复通路引发的肿瘤耐药性的产品；
    9)制备抑制由DNA损伤诱导的多聚ADP核糖链生成引发的肿瘤耐药性的产品；
    a、抑制CTD-2256P15.2基因的表达；
    b、抑制由CTD-2256P15.2基因编码的微肽PACMP的生物学功能；
    c、抑制含有PACMP的融合蛋白的生物学功能；
    d、抑制含有PACMP的复合物的生物学功能；
    所述CTD-2256P15.2基因的核苷酸序列为序列表中序列1或序列1的第138-272位。
  2. 根据权利要求1所述的应用，其特征在于：
    所述肿瘤为乳腺癌、卵巢癌、肺癌、肝癌、胃癌、结直肠癌、头颈癌、膀胱癌、宫颈癌、弥漫型B大细胞淋巴瘤、食管癌、胶质细胞瘤、胰腺癌、前列腺癌、黑色素瘤、胸腺瘤或子宫内膜癌。
  3. 根据权利要求1或2所述的应用，其特征在于：
    所述抑制剂为抑制CTD-2256P15.2基因的转录或翻译的物质、促进CTD-2256P15.2基因或微肽PACMP降解的物质或抑制微肽PACMP生物学功能的物质。
  4. 权利要求1-3任一项中的所述抑制剂和其他抗肿瘤物质在如下至少一种中的应用：
    1)制备肿瘤治疗产品；
    2)制备降低肿瘤细胞对化疗药物的耐药性的产品；
    3)制备提高肿瘤细胞对化疗药物的敏感性的产品；
    所述其他抗肿瘤物质为其他抗肿瘤药物或其他抗肿瘤治疗方法所需的试剂或仪器。
  5. 一种产品，其包括权利要求1-3任一项中的所述抑制剂，和，其他肿瘤治疗药物或其他抗肿瘤治疗方法所需的试剂或仪器；
    所述产品具有如下至少一种功能：
    1)肿瘤治疗；
    2)降低肿瘤细胞对化疗药物的耐药性；
    3)提高肿瘤细胞对化疗药物的敏感性。
  6. CTD-2256P15.2或其编码的微肽PACMP在作为治疗肿瘤试剂的作用靶点中的应用。
  7. CTD-2256P15.2基因作为标志物在制备评估肿瘤患者对化疗药物敏感性或预测肿瘤患者预后状态产品中的应用。
  8. 检测肿瘤组织中CTD-2256P15.2的表达量的物质在如下中的应用：
    1)制备预测肿瘤患者化疗后预后状态的产品；
    2)制备评估或辅助评估肿瘤患者对化疗药物敏感性的产品。
  9. 检测肿瘤组织中CTD-2256P15.2的表达量的物质和数据处理装置的应用，为1)-2)中的任一种：
    1)制备预测肿瘤患者化疗后预后状态的产品；
    2)制备评估或辅助评估肿瘤患者对化疗药物敏感性的产品；
    所述数据处理装置内设模块；所述模块具有如下(a1)和(a2)所示的功能：
    (a1)以肿瘤患者组成的待测群体的离体肿瘤组织为标本，测定每份标本中所述CTD-2256P15.2基因的表达量，然后根据基因表达量将所述待测群体分为低表达组和高表达组；
    (a2)按照如下标准确定来自于所述待测群体的待测患者的预后：
    所述低表达组中的待测患者化疗后的预后状态好于或候选好于所述高表达组中的待测患者；
    或，所述低表达组中的待测患者化疗后的预后总生存期长于或候选长于所述高表达组中的待测患者；
    或，所述低表达组中的待测患者化疗后的预后总生存率高于或候选高于所述高表达组中的待测患者；
    或，所述低表达组中的待测患者化疗后的预后无疾病进展生存期长于或候选长于所述高表达组中的待测患者；
    或，所述低表达组中的待测患者化疗后的预后无疾病进展生存率高于或候选高于所述高表达组中的待测患者；
    或，所述低表达组中的待测患者对化疗药物的敏感度高于或候选高于所述高表达组中的待测患者。
  10. 用于预测待测肿瘤患者化疗后预后的系统，包括检测肿瘤组织中CTD-2256P15.2的表达量的物质和权利要求9中的所述数据处理装置。
  11. 一种治疗或辅助治疗肿瘤的方法，包括如下步骤：单独使用权利要求1-3任一项中的所述抑制剂治疗肿瘤。
  12. 一种治疗或辅助治疗肿瘤的方法，包括如下步骤：权利要求1-3任一项中的所述抑制剂和其他肿瘤治疗药物配合使用，治疗肿瘤。
  13. 一种治疗或辅助治疗肿瘤的方法，包括如下步骤：使用权利要求1-3任一项中的所述抑制剂和其他肿瘤治疗方法配合使用，治疗肿瘤。
  14. 一种对肿瘤病人进行化疗药物治疗后预后状态辅助评估的方法，包括如下步骤：检测肿瘤病人的肿瘤组织中的CTD-2256P15.2的表达量；
    CTD-2256P15.2的表达量高的病人其预后状态差于或候选差于CTD-2256P15.2的表达量低的病人。
  15. 一种对肿瘤病人进行对化疗药物敏感性评估或辅助评估的方法，包括如下步骤：检测肿瘤病人的肿瘤组织中的CTD-2256P15.2的表达量；
    CTD-2256P15.2的表达量高的病人对化疗药物敏感性低于或候选低于CTD-2256P15.2的表达量低的病人。
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