CN114042160A

CN114042160A - CTD-2256P15.2 and application of encoded micro-peptide thereof as target in developing tumor treatment drugs

Info

Publication number: CN114042160A
Application number: CN202111346914.XA
Authority: CN
Inventors: 唐铁山; 郭彩霞; 周博; 张传超; 姚富文; 谷峰; 马勇杰
Original assignee: Institute of Zoology of CAS; Beijing Institute of Genomics of CAS; Tianjin Medical University Cancer Institute and Hospital
Current assignee: Institute of Zoology of CAS; Beijing Institute of Genomics of CAS; Tianjin Medical University Cancer Institute and Hospital
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-02-15
Anticipated expiration: 2041-11-15
Also published as: CN114042160B

Abstract

The invention discloses application of CTD-2256P15.2 and a coded micro-peptide thereof as a target point in developing a tumor treatment medicament. The invention provides the following uses of CTD-2256P15.2 inhibitors: 1) preparing a tumor treatment product; 2) preparing a product for reducing the drug resistance of tumor cells to chemotherapeutic drugs; 3) preparing a product for improving the sensitivity of tumor cells to chemotherapeutic drugs; the CTD-2256P15.2 or the coded micro-peptide PACMP inhibitor thereof provided by the invention can obviously inhibit the growth of tumor cells, increase the apoptosis of the tumor cells and reduce the tumor volume when acting on the tumor cells or tumor tissues, and has excellent anti-tumor effect.

Description

CTD-2256P15.2 and application of encoded micro-peptide thereof as target in developing tumor treatment drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of CTD-2256P15.2 and a coded micro-peptide thereof as a target spot in development of tumor treatment drugs.

Background

DNA damaging agents are commonly used in tumor therapy, such as radiation therapy and many chemotherapeutic drugs, mainly by inducing DNA damage in cancer cells, inhibiting cancer cell proliferation, inducing their death. However, tumor cells often resist the action of radiotherapy and chemotherapy drugs by changing their own DNA damage repair function, and generate tumor resistance (including primary and secondary resistance). PARP Inhibitors (PARPi) were the first drugs successfully approved for clinical use using the synthetic lethal concept and were shown to be highly effective in treating patients with BRCA1/2 mutated tumors. It is currently believed that PARPi inhibits the catalytic activity and single strand break repair of poly ADP polymerases such as PARP1, leading to DNA Double Strand Break (DSB) accumulation, or captures PARP1/2 on the DNA strand, interfering with its timely removal from the site of damage, thereby triggering replication stress and DSB damage. After PARPi administration to patients with tumors bearing mutations in BRCA1 or BRCA2, DSBs cannot be efficiently repaired due to inhibition of the DNA homologous recombination repair pathway, leading to cell death. However, similar to the widely used chemotherapeutic drugs in clinical practice, the use of PARPi also leads to the development of drug resistance, which is largely due to the restoration of the repair function of DNA damage, and the development of these drug resistances severely limits the clinical use and efficacy of PARPi. It is currently known that more than 40% of BRCA mutant ovarian cancer patients do not benefit from PAPRi treatment. Therefore, the key molecules for regulating and controlling DNA damage response, chemotherapeutic drugs and PARPi drug resistance are found, so that the method is not only beneficial to finding a prognostic biomarker, but also beneficial to finding a new target, developing a combination therapy and improving the treatment effect of the tumor.

Long-chain non-coding RNA (lncRNA) is a non-coding RNA with the length of more than 200nt in cells, plays an important role in regulation and control in various physiological and pathological processes, and the abnormal expression of the long-chain non-coding RNA is closely related to tumorigenesis, development and prognosis. Several lncrnas were found to modulate DNA damage repair processes, such as NORAD, DDSR1 and lnc BGL 3. However, these studies have focused on lncRNA as a molecular scaffold to bind specific proteins to regulate DNA repair, and little is known about lncRNA associated with DNA damage repair and tumor chemotherapy sensitivity relative to its diverse mechanisms of action and important regulatory functions. Recent years have shown that lncRNA can encode bioactive micro-peptide to regulate tumor growth, invasion and metastasis. However, no report has been made so far as to whether the lncRNA-encoded micro-peptide regulates DNA damage repair and tumor chemotherapy resistance.

Disclosure of Invention

An object of the present invention is to provide the use of CTD-2256P15.2 inhibitors.

The invention provides an inhibitor with any one of the following functions a-d, which is applied to at least one of the following 1) -9):

1) preparing a tumor treatment product;

2) preparing a product for reducing the drug resistance of tumor cells to chemotherapeutic drugs;

3) preparing a product for improving the sensitivity of tumor cells to chemotherapeutic drugs;

4) preparing a product for inhibiting DNA homologous recombination repair passages in tumor cells;

5) preparing a product for inhibiting a microhomology-mediated end-joining repair pathway in a tumor cell;

6) preparing a product for inhibiting generation of poly ADP ribose chains induced by DNA damage;

7) preparing a product for inhibiting tumor drug resistance caused by a DNA homologous recombination repair pathway;

8) preparing a product for inhibiting tumor drug resistance caused by a terminal connection repair path mediated by micro homology in tumor cells;

9) preparing a product for inhibiting tumor drug resistance caused by poly ADP ribose chain generation induced by DNA damage;

a. inhibiting the expression of CTD-2256P15.2 gene;

b. inhibits the biological function of the micro-peptide PACMP encoded by the CTD-2256P15.2 gene;

c. inhibiting a biological function of a fusion protein comprising PACMP;

d. inhibiting a biological function of a complex comprising a PACMP;

the nucleotide sequence of the CTD-2256P15.2 gene is shown as sequence 1 in the sequence table or 138 th-272 th position of sequence 1.

The amino acid sequence of the micro-peptide PACMP is a sequence 8 in a sequence table.

The tumor treatment product is a tumor treatment drug, and has the functions of inhibiting the proliferation of tumor cells or inducing the death of the tumor cells or inhibiting the growth of tumors.

Furthermore, in the tumor treatment drug, the CTD-2256P15.2 or the inhibitor of the encoded micro-peptide PACMP is used as the only effective component or one of the effective components.

In the above applications, the tumor includes, but is not limited to, breast cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, head and neck cancer, bladder cancer, cervical cancer, diffuse B large cell lymphoma, esophageal cancer, glioma, pancreatic cancer, prostate cancer, melanoma, thymoma, and endometrial cancer.

In the above application, the inhibitor is a molecule having an inhibitory effect on CTD-2256P15.2 or its encoded micro-peptide PACMP, and the inhibitory effect includes but is not limited to: inhibit the transcription or translation of CTD-2256P15.2, promote the degradation of CTD-2256P15.2 or PACMP and inhibit the function of PACMP.

The CTD-2256P15.2 or oligopeptide PACMP inhibitor can be siRNA, shRNA, antisense RNA, miRNA, gene knockout or knockdown CRISPR related plasmids and viral vectors, antibodies, polypeptides and small molecular compounds.

Specifically, the inhibitor is siRNA or shRNA targeting CTD-2256P15.2, or gRNA targeting PACMP coding region, or a vector expressing the above RNA;

the nucleotide sequence of the siRNA of the target CTD-2256P15.2 RNA is a sequence 2 or a sequence 3 in a sequence table;

the nucleotide sequence of shRNA of the targeting CTD-2256P15.2 RNA is sequence 4 or sequence 5 in the sequence table;

the nucleotide sequence of gRNA of the target PACMP coding region is sequence 6.

The function of the siRNA and shRNA for inhibiting the CTD-2256P15.2 provided by the invention is to target the RNA sequence of the CTD-2256P15.2 and reduce the RNA level thereof, and the function comprises the sequence and all siRNA and shRNA with similar functions.

The inhibitor and other antitumor substances (namely a novel antitumor drug combination scheme) are applied to at least one of the following substances:

1) preparing a tumor treatment product;

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 capable of causing damage to tumor cell DNA or inducing replication stress, and include but are not limited to: anthracyclines, camptothecin, PARP inhibitors, ATR inhibitors, CDK4/6 inhibitors, radiation therapy.

Furthermore, the anti-tumor drug is a chemotherapeutic drug.

The novel antineoplastic drug combination scheme can be any one of the following forms:

(1) the CTD-2256P15.2 inhibitor or the encoded micro-peptide PACMP inhibitor and other tumor treatment drugs or treatment methods are independently administered, the administration routes can be the same or different, and the inhibitor can be independently administered before, during and after the course of the other tumor treatment drugs or treatment methods.

(2) The CTD-2256P15.2 or the coded micro-peptide PACMP inhibitor and other tumor treatment medicines are prepared into a compound preparation, namely when the CTD-2256P15.2 or the coded micro-peptide PACMP inhibitor and other tumor treatment medicines are simultaneously administrated by the same administration route, the CTD-2256P15.2 and other tumor treatment medicines are prepared into the compound preparation.

It is another object of the present invention to provide a product comprising the above inhibitor, and, other agents or apparatus necessary for oncology therapy or other anti-oncology therapy methods;

the product has at least one of the following functions:

1) treating tumors;

2) reducing the drug resistance of the tumor cells to the chemotherapeutic drugs;

3) improve the sensitivity of the tumor cells to the chemotherapeutic drugs.

The application of the CTD-2256P15.2 or the encoded micro-peptide PACMP thereof in serving as a target of tumor treatment agents is also within the protection scope of the invention.

The CTD-2256P15.2 gene is used as a marker in the preparation of products for evaluating the sensitivity of tumor patients to chemotherapeutic drugs or predicting the prognosis state of tumor patients.

It is still another object of the present invention to provide use of a substance for detecting the expression level of CTD-2256P15.2 in a tumor tissue.

The substance for detecting the expression quantity of CTD-2256P15.2 in the tumor tissue provided by the invention is applied to the following steps:

1) preparing a product for predicting the prognosis state of a tumor patient after chemotherapy;

2) preparing a product for evaluating or assisting in evaluating the sensitivity of tumor patients to chemotherapeutic drugs.

The invention also provides a substance for detecting the expression quantity of CTD-2256P15.2 in tumor tissues and an application of a data processing device, which are any one of 1) to 2):

2) preparing a product for evaluating or assisting in evaluating the sensitivity of a tumor patient to chemotherapeutic drugs;

the data processing device is internally provided with a module; the module has the functions as shown in (a1) and (a 2):

(a1) taking in-vitro tumor tissues of a population to be detected consisting of tumor patients as samples, determining the expression quantity of the CTD-2256P15.2 gene in each sample, and then dividing the population to be detected into a low expression group and a high expression group according to the gene expression quantity;

(a2) determining the prognosis of a test patient from said test population according to the following criteria:

the prognosis state of the patients to be tested in the low expression group after chemotherapy is better than or candidate better than that of the patients to be tested in the high expression group;

or, the overall prognostic survival of the test patients in the low expression group after chemotherapy is longer or is candidate longer than that of the test patients in the high expression group;

or the prognosis total survival rate of the patients to be tested in the low expression group after chemotherapy is higher or the candidate is higher than that of the patients to be tested in the high expression group;

or, the prognosis disease progression free survival of the test patients in the low expression group after chemotherapy is longer or is candidate longer than the test patients in the high expression group;

or the survival rate of the prognosis disease-free progression after chemotherapy of the patients to be tested in the low expression group is higher than or is candidate to be higher than that of the patients to be tested in the high expression group;

or the sensitivity of the patients to be tested in the low expression group to the chemotherapeutic drugs is higher or the candidate is higher than that of the patients to be tested in the high expression group.

The invention also provides a system for predicting the prognosis of a patient with tumor to be detected after chemotherapy, which comprises a substance for detecting the expression quantity of CTD-2256P15.2 in tumor tissues and the data processing device.

The substance for detecting the expression quantity of the CTD-2256P15.2 in the tumor tissue is a probe or primer which is specifically combined with or amplifies the CTD-2256P 15.2.

The chemotherapy drugs adopted in the chemotherapy are tumor treatment drugs capable of causing DNA damage, and include but are not limited to anthracyclines, camptothecin or PARP inhibitors.

In the reagent for evaluating the sensitivity or prognosis of the tumor patient to the chemotherapeutic drugs, the expression of the CTD-2256P15.2 gene is used as one of the only detection indexes or effective detection indexes.

The invention also provides the following method:

the invention provides a method for treating or assisting in treating tumors, which comprises the following steps: the above inhibitors are used alone for treating tumors.

The invention provides a method for treating or assisting in treating tumors, which comprises the following steps: the inhibitor can be used in combination with other tumor therapeutic drugs for treating tumor.

The invention provides a method for treating or assisting in treating tumors, which comprises the following steps: the inhibitor is used in combination with other tumor treatment methods to treat tumors.

The invention provides a method for auxiliary evaluation of prognosis state of a tumor patient after chemotherapy drug treatment, which comprises the following steps: detecting the expression level of CTD-2256P15.2 in tumor tissues of a tumor patient;

patients with high expression of CTD-2256P15.2 have a worse prognosis or are candidate for worse than patients with low expression of CTD-2256P 15.2.

The invention provides a method for evaluating the sensitivity of a tumor patient to a chemotherapeutic drug or for assisting in evaluating the sensitivity of the tumor patient to the chemotherapeutic drug, which comprises the following steps of: detecting the expression level of CTD-2256P15.2 in tumor tissues of a tumor patient;

patients with high expression of CTD-2256P15.2 are less sensitive to chemotherapeutic drugs or are less candidate than patients with low expression of CTD-2256P 15.2.

The above-mentioned substance for detecting the expression level of CTD-2256P15.2 in tumor tissues is specifically a primer for fluorescent quantitative PCR in the examples.

The prognostic status is specifically expressed in disease progression free survival and/or overall survival, and specifically:

the overall survival time of the tumor patients corresponding to the tumor tissues with high expression level of the CTD-2256P15.2 gene after chemotherapy is smaller or is less than that of the tumor patients corresponding to the tumor tissues with low expression level of the CTD-2256P15.2 gene;

or, the tumor tissue corresponding to the tumor tissue with high expression of the CTD-2256P15.2 gene has a survival time without disease progression after chemotherapy smaller than or is less than that of the tumor tissue corresponding to the tumor tissue with low expression of the CTD-2256P15.2 gene.

The chemotherapy drugs are tumor treatment drugs capable of causing DNA damage, and include but are not limited to anthracyclines, camptothecin or PARP inhibitors;

further, the drug resistance is chemotherapy resistance.

CTD-2256P15.2 is highly expressed in various tumor types and can be induced to be expressed by various DNA damage chemotherapeutic drugs. CTD-2256P15.2 may encode a functional mini-peptide PACMP. The PACMP can competitively inhibit the combination of the latter and CtIP which is an important factor in a homologous recombination repair pathway and ubiquitination degradation by combining with a substrate adapter protein KLHL15 of ubiquitin ligase Cul3 on one hand, and promote PAR signal amplification by combining with a PAR chain induced by chemotherapy and DNA damage on the other hand, and the PAR signal amplification and the PAR chain synergistically promote the growth and the drug resistance of tumor cells. Therefore, the CTD-2256P15.2 or the encoded micro-peptide PACMP thereof are new targets for inhibiting tumor growth and enhancing the clinical treatment effect of tumors.

The CTD-2256P15.2 or the coded micro-peptide PACMP inhibitor thereof provided by the invention can obviously inhibit the growth of tumor cells, increase the apoptosis of the tumor cells and reduce the tumor volume when acting on the tumor cells or tumor tissues, and has excellent anti-tumor effect. According to the novel anti-tumor drug combination scheme provided by the invention, the CTD-2256P15.2 or the inhibitor of the coded micro-peptide PACMP and other anti-tumor drugs are used in a combined manner, so that the killing effect of the anti-tumor drugs on tumor cells can be obviously enhanced, the chemotherapy drug resistance of the tumor cells is reduced, and the clinical treatment effect of tumors is improved. CTD-2256P15.2 is highly expressed in chemotherapy-resistant tumor tissues and cell lines, and its high expression is significantly inversely correlated with disease progression-free survival and overall survival in tumor patients. The CTD2256P15.2 gene expression level provided by the invention can be used as an application of molecular indexes for predicting the sensitivity and prognosis of tumor patients to chemotherapy, and creates a new standard for effectively guiding clinical chemotherapy medication of tumor patients and evaluating treatment prognosis.

Drawings

FIG. 1 shows that CTD-2256P15.2 is highly expressed in the epirubicin-resistant breast cancer cell line MCF 7-EPI.

FIG. 2 shows that CTD-2256P15.2 expression is negatively correlated with prognosis in breast cancer patients.

FIG. 3 shows that the knock-down of CTD-2256P15.2 enhances the sensitivity of various tumor cells to epirubicin drugs.

FIG. 4 is a graph showing that knocking down CTD-2256P15.2 increases the sensitivity of tumor cells to various treatment regimens, including chemotherapy, targeted therapy and radiation therapy.

FIG. 5 shows that CTD-2256P15.2 encodes a mini-peptide.

FIG. 6 shows that CTD-2256P15.2 regulates the chemosensitivity of tumor cells by its encoded mini-peptide.

FIG. 7 shows that CTD-2256P15.2 or its encoded polypeptide can significantly inhibit tumor growth and enhance chemotherapeutic drug sensitivity.

FIG. 8 shows that the inhibition of CTD-2256P15.2 or its encoded mini-peptide can reduce the efficiency of homologous recombination repair and microhomologous end joining repair, reduce CtIP protein level and DNA damage induced PAR level.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Overall Survival (OS) in the following examples is defined as the time from enrollment to death or last follow-up due to any cause.

The overall survival rate in the following examples is defined as the probability that a patient will survive from a particular time point to a particular time.

Disease Progression Free Survival (PFS) in the examples below is defined as the time between the onset of treatment, until disease progression is observed or death occurs for any reason in a patient with a neoplastic disease.

The survival without disease progression in the following examples is defined as the probability that no disease progression is observed in a patient from a certain time point on follow-up to a certain time.

Example 1 Long non-coding RNA CTD-2256P15.2 and its use as a marker for the prediction of the prognosis status of chemotherapy

Discovery of single-chain non-coding RNA CTD-2256P15.2

The transcriptome sequencing is carried out on breast cancer tumor tissues sensitive and resistant to the anthracycline chemotherapeutic drug epirubicin, and the long-chain non-coding RNA CTD-2256P15.2 is identified to be highly expressed in the tumor tissues resistant to the epirubicin.

The nucleotide sequence of the long-chain non-coding RNA CTD-2256P15.2 is the sequence 1 in the sequence table.

Secondly, verifying the relation between CTD-2256P15.2 expression and tumor drug resistance

The method for detecting the drug resistance of the tumor cells to the epirubicin by using the CCK8 comprises the following steps:

MCF7-EPI cells (epirubicin-resistant cells obtained by increasing the epirubicin concentration stepwise (from 20nM to 500nM) during MCF7 cell culture) and control cell MCF7(ATCC Cat # HTB-22, RRID: CVCL-0031) in log phase were seeded into 96-well plates at 4000 cells per well, respectively. After 12h of culture, different concentrations of Epirubicin (EPI) were added to the culture system in each well. After an additional 24 hours of incubation, the medium was aspirated and fresh medium containing 10% (by volume) CCK8 reagent (DOJINDO, Cat # CK04) was added and incubated for 4 hours. Then, the absorbance of each well at 465nm is detected by a microplate reader, and a cell relative survival curve is drawn.

As shown in FIG. 1A, it can be seen that, as the concentration of EPI increases, the survival rate of the epirubicin-resistant cell line MCF7-EPI is higher than that of the cell line MCF7, i.e., MCF7-EPI is proved to be resistant to EPI.

Extracting total RNA of MCF7-EPI cells and MCF7 cell lines by using Trizol reagent, performing reverse transcription on the extracted RNA into cDNA by using reverse transcriptase, and detecting the RNA level of CTD-2256P15.2 by using real-time fluorescent quantitative PCR.

The primer for real-time fluorescent quantitative PCR detection is CTD-2256P 15.2: a forward primer: GACTTCTGCATTTGGCTGGAAGG, reverse primer: CTAACTCAGGGTATCGGAACCGA, respectively; internal reference GAPDH: a forward primer: GGAGCGAGATCCCTCCAAAAT, reverse primer: GGCTGTTGTCATACTTCTCATGG are provided.

As a result, as shown in FIG. 1B, it can be seen that the expression of CTD-2256P15.2 in MCF7-EPI is significantly higher than that of the control MCF7 cell.

Third, the relation between the expression of CTD-2256P15.2 and the sensitivity of tumor patients to chemotherapy

In order to further verify the relation between the expression of CTD-2256P15.2 and the chemotherapy sensitivity of tumor patients, 92 tumor tissues (from tumor hospital of Tianjin medical university) of breast cancer patients who received epirubicin-based chemotherapy were collected, wherein 48 patients had no tumor progression after chemotherapy and better prognosis, and the group had good prognosis; 44 patients had progressed following chemotherapy, and the prognosis was poor and was a poor prognosis group (tables 1 and 2).

Table 1 shows the results of the expression levels of tumor tissues in 44 patients with prognosis-poor breast cancer

In the above table, 1 in the recurrence status in column 3 indicates recurrence in the follow-up time of

column

2, and 0 indicates no recurrence or missed follow-up in the follow-up time of column 2; a1 in the death status in column 5 indicates death during the visit time in column 4 and a 0 indicates non-death or missed visit during the visit time in column 4.

Table 2 shows the results of the expression levels of tumor tissues in 48 patients with breast cancer in the prognosis group

column

The 92 breast cancer tumor tissue samples were formalin-fixed paraffin-embedded tissue sections, tumor tissue RNA was extracted with a nucleosin total RNA extraction kit, and the specific extraction procedure was according to the instructions of the kit. The extracted RNA was then reverse transcribed into cDNA using reverse transcriptase, and the expression of CTD-2256P15.2 in these tumor tissues was then detected using real-time fluorescent quantitative PCR.

The method of the fluorescence quantitative PCR technology is the same as the two real-time fluorescence quantitative PCR.

Patients with expression levels higher than the median expression level of all patients (median 1.15) were classified as high-expressing patients, and patients with expression levels lower than or equal to the median expression level of all patients were classified as low-expressing patients.

The results are shown in FIG. 2, wherein A is the expression level of CTD-2256P15.2 in patients with poor prognosis and good prognosis, B is survival without disease Progression (PFS), and C is Overall Survival (OS), and it can be seen that CTD-2256P15.2 is significantly higher in tumor tissue of patients with poor prognosis than in patient with good prognosis (FIG. 2A); by analyzing the prognosis of these tumor patients, it was found that the disease progression free survival (PFS, FIG. 2B, response in disease progression free survival) and overall survival (OS, FIG. 2C, response in overall survival) of patients with high expression of CTD-2256P15.2 in tumor tissues was shorter and less prognostic than those with low expression of CTD-2256P15.2, reflecting that patients with high expression of CTD-2256P15.2 were less sensitive to drugs, leading to disease progression.

Therefore, the detection of the expression quantity of the CTD-2256P15.2 gene in the tumor tissue can predict the sensitivity of the tumor patient to chemotherapeutic drugs; or detecting the expression level of CTD-2256P15.2 gene in tumor tissue to predict the prognosis state of tumor patient, especially the disease progression-free survival and/or overall survival;

the sensitivity of the tumor patient corresponding to the tumor tissue with high expression level of the CTD-2256P15.2 gene to chemotherapeutic drugs is lower than that of the tumor patient corresponding to the tumor tissue with low expression level of the CTD-2256P15.2 gene;

the prognosis state of a tumor patient corresponding to the tumor tissue with high expression level of the CTD-2256P15.2 gene after the treatment of the chemotherapeutic drug is worse than that of a tumor patient corresponding to the tumor tissue with low expression level of the CTD-2256P15.2 gene;

in particular to a tumor patient corresponding to a tumor tissue with high expression level of CTD-2256P15.2 gene, the overall survival time after chemotherapy is less than that of a tumor patient corresponding to a tumor tissue with low expression level of CTD-2256P15.2 gene;

or the disease progression-free survival time of the tumor patient corresponding to the tumor tissue with high expression level of the CTD-2256P15.2 gene after chemotherapy is smaller than that of the tumor patient corresponding to the tumor tissue with low expression level of the CTD-2256P15.2 gene.

Example 2 CTD-2256P15.2 as target for tumor therapy

Expression of CTD-2256P15.2 in various tumors

To verify that inhibition of CTD-2256P15.2 increases the sensitivity of various tumors to chemotherapeutic drugs, the expression of CTD-2256P15.2 in various tumors was first analyzed.

The transcriptome sequencing quantitative data of various tumors of TCGA is retrieved on GePIA website. The Ensembl ID of the CTD-2256P15.2 gene is ENSG00000259802, and the expression of CTD-2256P15.2 in various tumor type tissues can be obtained by searching the ID. BLCA: bladder urothelial cancer; BRCA: breast infiltrating cancer; CESC: squamous carcinoma of the cervix and adenocarcinoma; COAD: colon cancer; DLBC: diffuse large B-cell lymphoma; ESCA, esophageal cancer; GBM is glioblastoma multiforme; HNSC-head and neck squamous cell carcinoma; LGG, brain low-grade glioma; LUAD: lung adenocarcinoma; LUSC is squamous cell lung carcinoma; OV ovarian serous cystadenocarcinoma; PAAD pancreatic cancer; PRAD prostate cancer; READ rectal adenocarcinoma; STAD is gastric cancer; THYM thymus carcinoma; UCEC is endometrial cancer; UCS is uterine sarcoma.

The results are shown in FIG. 3A, TPM Transcripts Per Million; it can be seen that the expression of the CTD-2256P15.2 gene is higher in tumor tissues of a plurality of tumor types such as breast cancer, lung cancer, gastric cancer, ovarian cancer, etc. than in normal tissues.

Therefore, CTD-2256P15.2 can be selected as a therapeutic target in a number of tumors for the following experiments.

Application of CTD-2256P15.2 inhibitor siRNA to improvement of sensitivity of tumor cells to chemotherapy

1. Preparation of CTD-2256P15.2 inhibitor siRNA

In order to detect the regulation effect of CTD-2256P15.2 on the chemotherapy sensitivity of tumor cells, two specific siRNAs targeting CTD-2256P15.2 (CTD-2256P15.2 inhibitor siRNA) silnc15.2-1 and silnc15.2-2 are designed and synthesized according to the RNA sequence of CTD-2256P15.2, and the sequences are as follows:

silnc15.2-2, GCGGCUUCUGGAGGGACAA (SEQ ID NO: 2);

silnc15.2-1, GCAGAUGACCUAGCACAAA (SEQ ID NO: 3);

the sequence of control sirna (sinc) was: UUCUCCGAACGUGUCACGU are provided.

2. Transfection of siRNA and chemotherapy sensitivity experiments

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-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 (SK-OV-3Cat # HTB-77; RRID: CVCL _0532) and non-small cell lung cancer cells (CCL # 185: CCL-0023; RRID: CCL # 185 nM; CCL # 002CL) using lipofectamine RNAiMAX (Invitrogen, Cat # 13778150); specifically, knockdown was performed using a 3ml transfection system with a working siRNA concentration of 100nM in a 6cm dish (approximately 10^6 cells).

After 48 hours of transfection with different siRNAs, part of cells were trypsinized, centrifuged to collect cells, total RNA of the cells was extracted and reverse-transcribed into cDNA, and the level of CTD-2256P15.2 in the transfected cells was determined by fluorescent quantitative PCR.

Fluorescent quantitative PCR detection method example 1, results of different siRNA transfection MCF7 cells are shown in FIG. 3B, and it can be seen that silnc15.2-1 and silnc15.2-2 achieve CTD-2256P15.2 knock-down in tumor cells and reduce expression level thereof.

Meanwhile, after 24 hours of transfection, part of the cells are inoculated into a 96-well plate, after the cells adhere to the wall, different concentrations of epirubicin are added into a cell culture system for continuous treatment for 24 hours, and the survival condition of the cells is detected by a CCK8 method (the method is the same as the second method of the example 1).

The survival condition results of different tumor cells transfected with CTD-2256P15.2 inhibitor siRNA detected by the CCK8 method are shown in FIG. 3C-3I, and it can be seen that, compared with control siRNA, the CTD-2256P15.2 inhibitory siRNA knockdown of CTD-2256P155.2 in different tumor cells can significantly enhance the epirubicin sensitivity of the cells and reduce the survival rate of the tumor cells; the CTD-2256P15.2 is an effective target for enhancing the killing effect of tumor chemotherapy drugs and improving the clinical treatment effect.

Therefore, the siRNA for knocking out or inhibiting the expression of CTD-2256P15.2 can enhance the sensitivity of tumor cells to chemotherapeutic drugs and reduce the survival rate of the tumor cells treated by the chemotherapeutic drugs.

CTD-2256P15.2 inhibitory siRNAs and other therapies

Synthetic control siRNA (siNC), specific silnc15.2-1 targeting CTD-2256P15.2 and silnc15.2-2 were transfected into MCF7 cells using lipofectamine RNAIMAX (Invitrogen, Cat #13778150) according to the second protocol above, giving a working siRNA concentration of 100 nM.

After 24 hours of transfection, the transfected cells were inoculated into a 96-well plate, after the cells adhered to the wall, different concentrations of Camptothecin (Camptothecin, CPT) (Sigma, Cat # C9911), ATR inhibitor (VE-822) (Selleck, Cat # S7102) or CDK4/6 inhibitor (Palbociclib, Selleck, Cat # S1116) were added to the culture system of some cells, and after the treatment was continued for 48 hours, the survival of the cells was measured by using CCK8 reagent, which was the same as in example 1.

The results are shown in FIGS. 4A-4C, wherein A, B and C are siRNA + Camptothecin (CPT), siRNA + ATR inhibitor (VE-822) and siRNA + CDK4/6 inhibitor (Palbociclib), respectively, and it can be seen that the CTD-2256P15.2 inhibitory siRNA can significantly increase the sensitivity of MCF7 cells to tumor therapeutic drugs such as Camptothecin (CPT), ATR inhibitor (VE-822) and CDK4/6 inhibitor (Palbociclib) after inhibiting the expression of CTD-2256P 15.2.

Therefore, the results show that the combined action of CTD-2256P15.2 inhibitory siRNA and other chemoradiotherapy schemes or chemotherapeutics can remarkably enhance the killing effect on tumor cells, and a brand new strategy is provided for improving the tumor treatment effect. Namely, the CTD-2256P15.2 inhibitory siRNA can enhance the killing effect of the antitumor drug on tumor cells and reduce the chemotherapy drug resistance of the tumor cells.

Application of CTD-2256P15.2 inhibitor shRNA in improving sensitivity of tumor cells to chemotherapy

The expression level of CTD-2256P15.2 is reduced by shRNA technology, and the survival condition of tumor cells after Olaparib or X-ray treatment is detected by a clone formation experiment. The method comprises the following specific steps:

1. designing and synthesizing two shRNA coding sequences for targeting CTD-2256P15.2, wherein shlnc 15.2-2: GCGGCTTCTGGAGGGACAAAGCTCGAGCTTTGTCCCTCCAGAAGCCGC (SEQ ID NO: 4); shlnc 15.2-1: GCAGATGACCTAGCACAAATACTCGAGTATTTGTGCTAGGTCATCTGC (SEQ ID NO: 5).

shNC sequence: TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA

The plasmid was ligated to pLKO vector (Addgene Cat #10878https:// www.addgene.org/protocols/pLKO /), respectively, to obtain recombinant pLKO plasmid for expression of shRNA.

The recombinant pLKO-shlnc15.2-2 plasmid is a vector obtained by replacing the encoding gene of shlnc15.2-2 shown in a sequence 4 between AgeI and EcoRI sites of a pLKO vector, and the vector expresses shlnc15.2-2 (RNA encoded by the sequence 4).

The recombinant pLKO-shlnc15.2-1 plasmid is a vector obtained by replacing the encoding gene of shlnc15.2-1 shown in a sequence 5 between AgeI and EcoRI sites of a pLKO vector, and the vector expresses shlnc15.2-1 (RNA encoded by the sequence 5).

The recombinant pLKO-shNC plasmid is a vector obtained by replacing the encoding gene of shNC with AgeI and EcoRI sites of a pLKO vector.

2. Lentivirus packaging and infection

293T cells (Cat # CRL-3216; RRID: CVCL _0063) in the logarithmic growth phase were seeded one day before transfection in a 10cm dish to give a cell density of about 50%, and plasmid transfection was carried out after overnight culture.

The medium was changed to fresh medium without antibiotics and placed at 37 degrees before transfection. Mu.g plasmid (pLKO-shlnc 15.2-1 or pLKO-shlnc15.2-2 as described above and the virus packaging plasmid pRSV-Rev (Addgene ID 12253), pMD2.G (Addgene ID 12259), pMDLg/pRRE (Addgene ID 12251) (ratio 2:1:1:1) were transferred into 293T cells according to the protocol for the vigoFect transfection reagent, after 24 hours of transfection, the medium was discarded, 8ml of fresh complete medium was added and the culture continued, and after 48 hours, the cell culture supernatant contained the corresponding lentivirus.

MCF7 cells were seeded one day in advance into 6cm dishes at a density of 50% for lentivirus infection. The next day of infection, 293T culture supernatants were collected and filtered through 0.45 μ M filters into clean centrifuge tubes. The filtered virus was mixed with an equal volume of fresh medium and polybrene at a final concentration of 10. mu.g/mL was added, mixed well and added to MCF7 cells, and the solution was changed 8 hours after infection. After culturing for 48 hours, 1mg/ml puromycin is added for screening, and when the cells can stably grow in puromycin and the good knockdown effect can be detected by qPCR, the cells are considered to obtain MCF7 cells which stably express shRNA (shCTD-2256P15.2) -1/2 targeting CTD-2256P15.2 and are named as MCF7 shRNA-1 and MCF7 shRNA-2.

Cells into which the recombinant pLKO-shNC plasmid was transferred were designated as MCF7 shNC.

The qPCR detection method described above is as follows: extracting total RNA of the cells, performing reverse transcription on the RNA into cDNA by using reverse transcriptase, and detecting the content of CTD-2256P15.2 by fluorescent quantitative PCR (primers as before), wherein the result is shown in FIG. 4D, and it can be seen that shlnc15.2-1 and shlnc15.2-2 can realize the knockout of CTD-2256P15.2 and reduce the expression thereof.

3. Function(s)

MCF7 shRNA-1 or MCF7 shRNA-2 obtained in the above 2 and MCF7 shNC (control cells) were inoculated into 6cm dishes, respectively.

After cell attachment, about 500 cells were treated with varying concentrations of the PARP inhibitor Olaparib (Olaparib) for 24 hours, the medium was discarded, the cells were washed 2 times with PBS, fresh complete medium was added, and the cells were placed in a 37-degree incubator for further 14 days. The culture medium was discarded, the cells were rinsed gently with water 2 times, the culture dish was dried at room temperature, the number of macroscopic cell clones was counted, the clone survival ratio was calculated according to the formula (survival ratio: the number of clones formed under different treatment conditions divided by the number of clones formed under different treatment conditions of the same group), and a clone survival curve was plotted.

Or irradiating the part of the cells with 500 adherent cells with different doses of X-rays to cause DNA damage, continuously culturing for 14 days, counting the number of cell clones, and drawing a clone survival curve.

The results are shown in fig. 4E and 4F, and the reduction of the expression of CTD-2256P15.2 in MCF7 cells by shRNA can significantly enhance the killing effect of PARP inhibitors and X-rays on cells, reduce the survival of tumor cells, and increase the sensitivity of cells to PARP inhibitor chemotherapy and radiotherapy.

The results show that the combined action of the CTD-2256P15.2 inhibitor shRNA and other chemoradiotherapy schemes or chemotherapeutics can obviously enhance the killing effect on tumor cells, and a brand new strategy is provided for improving the tumor treatment effect. Namely, the CTD-2256P15.2 inhibitor shRNA can kill tumor cells with strong antitumor drugs or tumor treatment methods and reduce the chemotherapy drug resistance of the tumor cells.

Example 3, CTD-2256P15.2 encoded micropeptides and methods for modulating chemotherapeutic sensitivity in tumor cells

One, CTD-2256P15.2 coded micro-peptide

According to biological information prediction, the open reading frame 1(ORF1, namely 138 th-272 th position of the sequence 1) of CTD-2256P15.2 possibly encodes a mini-peptide with the amino acid length of 44 amino acids, and the amino acid sequence of the mini-peptide is MAASGGTKKAQSGGRRLREPSSRPSRRARQRPRRGALRKAGRFL (sequence 8).

In order to verify the encoding performance of the CTD-2256P15.2, a sequence containing an SBP-FLAG tag is knocked in at the end (before a stop codon) of the 3' end of the CTD-2256P15.2 gene ORF1 of U2OS cells by using a CRISPR-Cas9 technology. The monoclonal cell inserted successfully is obtained by picking the monoclonal cell and performing PCR sequencing identification.

The specific method comprises the following steps:

1) designing and synthesizing a DNA sequence specifically targeting the end of ORF 13': CGGCGTGCACTGTCGGTCGGCGG (SEQ ID NO: 6), and this DNA was cloned into a pX330 vector for transcription into a gRNA.

The recombinant vector pX330-gRNA is a vector obtained by inserting a DNA molecule represented by CGGCGTGCACTGTCGGTCGGCGG (SEQ ID NO: 6) between the Bbs1 sites of the pX330 vector (Addgene ID 42230), and expresses a gRNA (gRNA encoded by SEQ ID NO: 6).

2) Design and synthesis of donor DNA:

the donor DNA is a left homology arm-sequence containing a label (SBP-FLAG-P2A-puromycin) -right homology arm, and the nucleotide sequence is a sequence 7.

3) Knock-in

The donor DNA and pX330-gRNA plasmid were electroporated at 135V using a Pulse Generator-CUY21EDDID 2 electrotransfer apparatus into U2OS cells (ATCC, Cat # HTB-96; RRID: CVCL-0042). After 48 hours, 1mg/ml puromycin was added for selection, and then the cells grown after puromycin selection were monoclonally inoculated. After 2 weeks, monoclonal cells were picked and expanded, genomic DNA was extracted, and successful knock-in positive clone U2OS-KI cells were identified using PCR amplification and sequencing.

The primers used for the PCR amplification are: a forward primer: AGAGGCTGACAGAAAGCGAG, reverse primer: CTAACTCAGGGTATCGGAACCGA, an approximately 1580bp fragment was obtained and a positive clone containing the tap-in tag sequence, which was successful for tap-in, was sequenced and designated U2OS-KI cells, which express the fusion protein ORF1-SBP-FLAG-P2A (the nucleic acid encoding this fusion protein is sequence 10, which is a sequence obtained by fusing the 3' end of the first open reading frame of CTD-2256P15.2 (before the stop codon) to SBP-FLAG-P2A-puromycin, and upon protein translation, the P2A sequence was self-cleaving, thus obtaining the ORF1-SBP-FLAG-P2A fusion protein).

4) shCTD-2256P15.2 cells

shRNA technology for knocking down CTD-2256P15.2 in U2OS-KI cells obtained in 3) above, the method was as described in example 2, and a cell obtained by transfecting U2OS-KI cells with lentivirus using a recombinant pLKO-shlnc15.2-1 plasmid was designated as U2OS-KI-shlnc 15.2.

The recombinant pLKO-shNC plasmid is adopted to transfect U2OS-KI cells by slow virus, and the obtained cells are named as U2 OS-KI-shNC.

5) Detecting expression

A. Endogenous expression of ORF1 was verified in U2OS-KI control cells. The specific method comprises the following steps:

(1) immunofluorescence: logarithmic phase U2OS cells (control cells), U2OS-KI-shlnc15.2 and U2OS-KI-shNC cells were seeded onto slides at 60-80% density. The following day, the medium was aspirated, the cells were washed twice with PBS, and the cells were fixed for 20 minutes at room temperature by adding 4% paraformaldehyde solution. The fixative was discarded and the cells were washed 3 times with PBS. Adding 0.5% Triton X-100 in PBS, treating the cells at room temperature for 10 min, discarding the Triton solution, washing the cells with PBS 3 times, adding 5% BSA in PBS, and blocking at room temperature for 1 hr. The cells were then incubated sequentially with primary anti-SBP-tag antibody (Santa Cruz, Cat # sc-101595; RRID: AB-1128239), fluorescent secondary antibody (Life Technologies, Cat # A11029; RRID: AB-138404), and finally mounted with DAPI-containing blocking agent.

The expression of ORF1 fusion protein was detected microscopically.

As shown in FIG. 5A, WT was a U2OS cell, KI-shlnc15.2 was a U2OS-KI-shlnc15.2 cell, and KI-shNC was a U2OS-KI-shNC cell, and it was found that, in the U2OS-KI-shNC cell, ORF1 was able to initiate translation and express the ORF1-SBP-FLAG-P2A fusion protein, while the lnc 15.2-knocked-down cell had no expression of the fusion protein in U2OS-KI-shlnc 15.2.

(2) And (3) immunoprecipitation: collect 4 x 10⁶In the logarithmic growth phase, U2OS, U2OS-KI-shNC and U2OS-KI-shlnc15.2 cells were lysed with a lysis buffer containing 1% Triton and centrifuged, and anti-FLAG beads were added to the obtained cell lysate and incubated at 4 ℃ for 2 hours with rotation. After centrifugation to remove the supernatant and washing the beads with lysis buffer, 2 XSDS sample buffer was added and the mixture was boiled at 95 ℃ for 10 minutes. And detecting the expression condition of the ORF1 fusion protein by using a western blot technology.

The results are shown in fig. 5B, ORF1 was able to initiate translation in U2OS-KI-shNC, expressing ORF1-SBP-FLAG-P2A fusion protein, sh15.2 knockdown cells U2OS-KI-shlnc15.2 with significantly reduced expression of ORF1-SBP-FLAG-P2A compared to U2OS-KI-shNC cells.

These results indicate that ORF1 of CTD-2256P15.2 encodes a oligopeptide of 44 amino acids in length.

II, CTD-2256P15.2 is used for regulating and controlling the chemotherapy sensitivity of tumor cells through encoded micro-peptide

To verify the regulatory effect of the encoded micro-peptide of CTD-2256P15.2 on the chemotherapy sensitivity of tumor cells, the complementation expression of the CTD-2256P15.2 full-length wild type (FL) and the full-length start codon mutation of ORF1 (FL) resistant to shCTD-2256P15.2 in MCF7 and U2OS cells stably knocked down by CTD-2256P15.2 and the complementation of ORF1 fusion protein tested the sensitivity of the cells to epirubicin.

The specific method comprises the following steps:

1. construction of CTD-2256P15.2 stably knocked-down MCF7 and U2OS cells

CTD-2256P15.2 stably knockdown MCF7 and U2OS cells were constructed using shRNA technology, as detailed in example 2. Except that MCF7 and U2OS cells are adopted as cells, and pLKO-shlnc15.2-1 plasmid is adopted as recombinant plasmid, so that MCF7 cells stably expressing shRNA (shCTD-2256P15.2) -1 targeting CTD-2256P15.2 and U2OS cells stably expressing shRNA (shCTD-2256P15.2) -1 targeting CTD-2256P15.2 are obtained.

And (3) knocking out the recombinant pLKO-shNC plasmid as a control to obtain MCF7 cells for expressing shNC in a control mode and U2OS cells for expressing shNC in a control mode.

2. Construction of anaplerotic cell lines

The method comprises the following specific steps:

1) the Full Length (FL) of CTD-2256P15.2 wild-type Full Length (FL) resistant to shRNA, the full length (FL x) of ORF1 start codon mutation (ATG mutated to ATT, unable to start encoding the mini-peptide any more), and ORF1 fusion protein (ORF 1C-terminal fused SBP-FLAG tag) were ligated to a pNL lentiviral expression vector (pNL-EGFP/CMV/WPREdU3, addge, #17579), respectively, and recombinant plasmids were obtained by screening, as follows:

the recombinant plasmid FL is a vector obtained by cloning a gene sequence (sequence 1) of CTD-2256P15.2 between the Nhe1 enzyme cutting sites and the EcoR1 enzyme cutting sites of a pNL lentiviral expression vector;

the recombinant plasmid FL is a vector obtained by cloning an ORF1 initiation codon mutant sequence (the sequence of the ATG of the sequence shown in the 138 th and 141 th sites of the sequence 1 is mutated into ATT, namely the initiation codon ATG of the No. 1 open reading frame is mutated into ATT) of CTD-2256P15.2 between the Nhe1 enzyme cutting sites and the EcoR1 enzyme cutting sites of a pNL lentivirus expression vector;

the recombinant plasmid ORF1 is a vector obtained by cloning an encoding gene of ORF1-SBP-FLAG fusion protein (the sequence of the encoding gene is sequence 9, namely the sequence 1, the 138 th-269 th site + SBP-FLAG nucleic acid sequence) between the Nhe1 enzyme cutting sites and the EcoR1 enzyme cutting sites of a pNL lentivirus expression vector;

2) transfecting the recombinant plasmids and the packaging plasmids into 293T cells respectively by a lentivirus packaging and infection method (the method is the same as 2 of the fourth embodiment 2) to obtain a culture supernatant containing virus particles; then infecting MCF7 cell stably expressing shRNA (shCTD-2256P15.2) -1 targeting CTD-2256P15.2 and U2OS cell stably expressing shRNA (shCTD-2256P15.2) -1 targeting CTD-2256P15.2 with lentivirus respectively, so as to obtain the stable cell for replenishing, MCF7 cells (transferred recombinant plasmid ORF1) of ORF1 anaplerotic shRNA (shCTD-2256P15.2) -1, U2OS cells (transferred recombinant plasmid ORF1) of ORF1 anaplerotic shRNA (shCTD-2256P15.2) -1, MCF7 cells (transferred recombinant plasmid FL) of Fl anaplerotic shRNA (shCTD-2256P15.2) -1, U2OS cells (transferred recombinant plasmid FL) of Fl anaplerotic shRNA (shCTD-2256P15.2) -1, MCF7 cells (transferred recombinant FL) of Fl anaplerotic shRNA (shCTD-2256P15.2) -1 and U2OS cells (transferred recombinant plasmid FL) of Fl anaplerotic shRNA (shCTD-2256P15.2) -1 are respectively named.

The above cells were seeded in a 96-well plate, and the adherent cells were treated with 1. mu.M epirubicin for 24 hours, and the survival rate of the cells was examined using CCK8 reagent in the same manner as in example 1. MCF7 cells expressing shNC under control, U2OS cells expressing shNC under control, MCF7 cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2) -1 and U2OS cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2) -1 were used as controls.

The results are shown in FIG. 6, where A is MCF7 cells and B is U2OS cells; shNC means a cell expressing shNC, shlnc15.2 means a cell stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2) -1, shlnc15.2+ ORF1 means a cell stably expressing ORF1 anaplerotic shRNA (shCTD-2256P15.2) -1, shlnc15.2+ FL means a cell of FL anaplerotic shRNA (shCTD-2256P15.2) -1, it can be seen that knocking down CTD-2256P15.2 increases the sensitivity of tumor cells to epirubicin, anaplerotic expression of wild type (ORF1) and 1 fusion protein (FL) reduces the sensitivity of cells to epirubicin, whereas the full length of the anaplerotic expression of 1 mutation (FL) cannot be restored.

The results show that CTD-2256P15.2 regulates the chemotherapy sensitivity of tumor cells through its encoded micro-peptide.

Thirdly, verifying the regulation and control effect of CTD-2256P15.2 or the encoded micro-peptide on the sensitivity of tumor chemotherapy at the animal level

A triple-negative breast cancer cell line MDA-MB-231 with CTD-2256P15.2 stable knock-down and wild type full-length or ORF1 mutant full-length complementation is constructed, and a certain amount of cells are inoculated under the mammary fat pad of a female nude mouse. When the transplanted tumor grows to a certain size, the mice are respectively treated with normal saline and epirubicin, and the size of the tumor is measured and counted. At the end of the experiment, mice were sacrificed and tumors dissected, photographed and tumor weights measured.

The method comprises the following specific steps:

1) the construction of stably knockdown MDA-MB-231 cells for CTD-2256P15.2 using shRNA, and the construction of full-length mutant (FL) -recouped MDA-MB-231 cells for CTD-2256P15.2 wild-type full-length (FL) or ORF1 start codon mutation (mutation from ATG to ATT) by lentiviral infection in knockdown cells, is as follows:

referring to example 2, recombinant pLKO-shlnc15.2-1 plasmid was transferred into MDA-MB-231 cell line to obtain MDA-MB-231 cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2) -1;

and (3) transfecting the recombinant pLKO-shNC plasmid as a control to obtain a control-knocked-down MDA-MB-231 cell expressing shNC.

Referring to 2 of the second example, the wild type lnc15.2 Full Length (FL) and the mutant lnc15.2 Full Length (FL) were transformed into MDA-MB-231 cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2) -1, respectively, to obtain MDA-MB-231 cells of FL-anaplemented shRNA (shCTD-2256P15.2) -1 and MDA-MB-231 cells of FL-anaplemented shRNA (shCTD-2256P15.2) -1.

2) Will be 4X 10⁶The cells in logarithmic growth phase were mixed with an equal volume of matrigel and inoculated under the mammary fat pad of female BALB/c nude mice. When the length of the tumor reaches 5mm, the mice are randomly divided into two groups, and 5mg/kg of epirubicin or physiological saline with the same volume is respectively injected into the abdominal cavity. The medicine is administrated once every 4-5 days, the tumor volume is measured every other day, and a tumor relative growth curve is drawn. Mice were sacrificed on day 11 after the initial dose, tumors were dissected out, photographed and tumor weights were measured. MDA-MB-231 cells expressing shNC and MDA-MB-231 cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2) -1 were used as controls.

The results are shown in FIG. 7, where A-C are (A) the growth of tumor tissue in the case of epirubicin treatment or no treatment of tumor cells in each transplanted group, (B) the weight of the transplanted tumor in each collected group, and (C) photographs of the transplanted tumor in each collected group, respectively. 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 shlnc5.2 MDA-MB-231 cells, shlnc15.2+ FL means complementation of mutant full length lnc15.2 in shlnc5.2 MDA-MB-231 cells, it can be seen from tumor volume, tumor size or tumor size that knocking down CTD-2256P15.2 significantly inhibits growth and increases sensitivity to epirubicin, that complementation of wild type but not mutant can restore growth and sensitivity to epirubicin.

The result shows that the CTD-2256P15.2 inhibitor or the encoded micro-peptide thereof has excellent clinical application potential, the tumor growth can be reduced by single inhibition, and the combined application with other chemotherapeutic drugs can enhance the killing effect of the chemotherapeutic drugs and improve the clinical treatment effect.

Fourthly, the action mechanism of CTD-2256P15.2 or the encoded micro-peptide for regulating the growth of the tumor and the sensitivity to the chemotherapeutic drugs is researched

1) DNA homologous recombination repair (HR) efficiency and microhomology-mediated end-linking (MMEJ) assay

DR-U2OS cells (described in Xia, B., Sheng, Q., Nakanishi, K., Ohashi, A., Wu, J., Christ, N., Liu, X., Jasin, M., Couch, F.J., and Livingston, D.M (2006). Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2.mol Cell 22,719 729.) and U2OS EGFP-MMEJ cells (described in Wang, H., Shao, Z., Shi, L.Z., Hwang, P.Y., Truong, L.N., Benz, M.W., Chemn, D.J., C., C.and S.sub.2, DR-U2-7, DR-U2N., DR-2, D.W., C., and S.2, S.sub.7, S.sub.2, S.2, DNA, siNC-knockout DR-U2OS cells, silnc 15.2-1-knockdown MMEJ cells, silnc 15.2-2-knockdown MMEJ cells, and siNC-knockout MMEJ cells.

The positive controls were introduced siBRCA1(UAUAAGACCUCUGGCAUGAAU) and siCtIP (GCUAAAACAGGAACGAAAUC).

And (3) sorting GFP positive cells by using a flow cytometer, and counting the GFP positive cells and the total cells respectively, wherein the proportion of the positive cells to the total cells is the DNA homologous recombination repair (HR) efficiency or the microhomologous mediated end-linking (MMEJ) efficiency.

As shown in FIGS. 8A (DR-U2OS cells) and 8B (U2OS EGFP-MMEJ cells), the efficiency of DNA homologous recombination repair (HR) and the efficiency of microhomology-mediated end-linking (MMEJ) were significantly reduced after knockdown of CTD-2256P15.2 by siRNA.

2) Protein content detection of HR (human HR) repair key factor CtIP (CtIP)

The two prepared MCF7 cells stably expressing shRNA (shCTD-2256P15.2) -1 of targeting CTD-2256P15.2, MCF7 cells (transferred into recombinant plasmid ORF1) of ORF1 anaplerotic shRNA (shCTD-2256P15.2) -1 and MCF7 cells (transferred into recombinant plasmid FL) of Fl anaplerotic shRNA (shCTD-2256P15.2) -1 are collected in logarithmic growth phase, and the protein level of CtIP in the cells is detected by a western blot method.

The result is shown in fig. 8C, after the shRNA is used for inhibiting the expression of CTD-2256P15.2, the protein content of the key HR repair factor CtIP in the MCF7 cell is significantly reduced, and the protein level of the CtIP can be recovered by complementing the full length of the expressed wild type instead of the full length of the ORF1 mutant.

3) PAR detection

And respectively transferring the pNL lentivirus empty expression vector (namely the pNL lentivirus expression vector), the recombinant plasmid FL and the recombinant plasmid FL into silnc15.2-1 cells for knocking down the MCF7 to obtain the MCF7 cells for complementing the silnc15.2-1 by the pNL lentivirus empty expression vector, the MCF7 cells for complementing the silnc15.2-1 by the FL and the MCF7 cells for complementing the silnc15.2-1 by the FL.

The cells in the logarithmic growth phase were seeded in a 6-well plate so that the cell density was about 70%. The next day, the medium was discarded and used with a medium containing 400. mu. M H₂O₂The cells were treated for 5 minutes at 37 degrees, the medium was discarded, the cells were washed twice with PBS, and 1 XSDS sample buffer was added to lyse the cells. The intracellular PAR levels were measured by western blot. To be free of H₂O₂The serum-free medium of (1) was used as a control.

The results are shown in FIG. 8D, with sinC being sinC-treated MCF7 cells, silnc15.2 being sinnc 15.2-1 treated MCF7 cells, and-pNL lentivirus empty expression vector complemented by silnc15.2-1 MCF7 cells, FL being FL complemented by silnc15.2-1 MCF7 cells, and FL being FL complemented by silnc15.2-1 MCF7 cells; it can be seen that the reduction of CTD-2256P15.2 can significantly inhibit the generation of poly-ADP ribose (PAR) modified chain induced by DNA damage, the full-length wild type can restore PAR generation, and the full-length ORF1 mutant can not restore PAR (FIG. 8D).

The results show that CtIP is a key factor of the normal initial DNA homologous recombination repair and the microhomology-mediated terminal link repair function of cells, and two DNA damage repair channels are damaged after the CtIP is deleted. The effect of inhibiting tumor growth can be achieved through the synthetic lethal effect by inhibiting CtIP and/or PAR caused by CTD-2256P15.2 or encoded micro-peptide PACMP.

SEQUENCE LISTING

<110> animal institute of Chinese academy of sciences Beijing genome institute of Chinese academy of sciences (national center for biological information) Tianjin City tumor hospital (Tianjin medical university tumor hospital)

<120> CTD-2256P15.2 and application of encoded micro-peptide thereof as target in developing tumor treatment drugs

<160>10

<170> PatentIn version 3.5

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tcaggagctg agctttgatg gcggcttctg gagggacaaa gaaggcgcag agcggtggga 180

ggaggctgcg agaacctagc tcccgcccca gccgacgtgc gagacaaagg ccccggcgcg 240

gggctcttcg gaaggccggg aggttcctgt gacaatgcga gtctgcgccc cacgctcccg 300

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ggagcagtct ttccagcctc gaaaaatgtt taaacaatat gcagatgacc tagcacaaat 780

aaagaatgct agcaaccgct gttatgtgtg agcggtggaa ttgggagggc tcttccccca 840

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a 901

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gggcgcggcg aatcctcgcg tgcgccaggc cgcctggggt agtcgccgag actgcgccgg 240

ccagcggggc cgaaacctaa tcaggagctg agctttgatg gcggcttctg gagggacaaa 300

gaaggcgcag agcggtggga ggaggctgcg agaacctagc tcccgcccca gccgacgtgc 360

gagacaaagg ccccggcgcg gggctcttcg gaaggccggg aggttcctgc ggcgcggggc 420

tcttcggaag gccgggaggt tcctgggcag cggcatggac gagaagacca ccggctggcg 480

gggcggccac gtggtggagg gcctggccgg cgagctggag cagctgcggg ccaggctgga 540

gcaccaccct cagggccagc gggaggatta caaggatgac gacgataagg gaagcggagc 600

tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctggatc 660

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ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga 840

catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga 900

gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg 960

ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga 1020

gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg 1080

cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct 1140

ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc 1200

cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg 1260

acaatgcgag tctgcgcccc acgctcccgc accgtcaatg cgagtctgcg ccccacgctc 1320

ccgcaccgtg atgccgaggc caaatggcta gagtgggccc aggcatcagc atttctgttt 1380

ggcagccggg cagttgtggt gcacgttttg aggaactact aaagcctgcc tgtgatgaaa 1440

aaggcaaaag ctgacttcac caaaattact cccagggaga cttctgcatt tggctggaag 1500

gacatttgag taaaccgctg aggctggtgg ttgaaacata atcctctaag ggagatcggt 1560

tccgataccc tgagttag 1578

<210> 8

<211> 44

<212> PRT

<213> Artificial sequence

<400> 8

Met Ala Ala Ser Gly Gly Thr Lys Lys Ala Gln Ser Gly Gly Arg Arg

1 5 10 15

Leu Arg Glu Pro Ser Ser Arg Pro Ser Arg Arg Ala Arg Gln Arg Pro

20 25 30

Arg Arg Gly Ala Leu Arg Lys Ala Gly Arg Phe Leu

35 40

<210> 9

<211> 270

<212> DNA

<213> Artificial sequence

<400> 9

atggcggctt ctggagggac aaagaaggcg cagagcggtg ggaggaggct gcgagaacct 60

agctcccgcc ccagccgacg tgcgagacaa aggccccggc gcggggctct tcggaaggcc 120

gggaggttcc tggacgagaa gaccaccggc tggcggggcg gccacgtggt ggagggcctg 180

gccggcgagc tggagcagct gcgggccagg ctggagcacc accctcaggg ccagcgggag 240

cccgattaca aggatgacga cgataagtaa 270

<210> 10

<211> 339

<212> DNA

<213> Artificial sequence

<400> 10

atggcggctt ctggagggac aaagaaggcg cagagcggtg ggaggaggct gcgagaacct 60

agctcccgcc ccagccgacg tgcgagacaa aggccccggc gcggggctct tcggaaggcc 120

gggaggttcc tgggcagcgg catggacgag aagaccaccg gctggcgggg cggccacgtg 180

gtggagggcc tggccggcga gctggagcag ctgcgggcca ggctggagca ccaccctcag 240

ggccagcggg aggattacaa ggatgacgac gataagggaa gcggagctac taacttcagc 300

ctgctgaagc aggctggaga cgtggaggag aaccctgga 339

Claims

1. Use of an inhibitor having any one of the following functions a-d in at least one of 1) to 9) as follows:

1) preparing a tumor treatment product;

4) inhibiting DNA homologous recombination repair pathways in tumor cells;

5) inhibiting a microhomology-mediated end-joining repair pathway in a tumor cell;

6) inhibiting generation of poly ADP ribose chain induced by DNA damage;

a. inhibiting the expression of CTD-2256P15.2 gene;

c. inhibiting a biological function of a fusion protein comprising PACMP;

d. inhibiting a biological function of a complex comprising a PACMP;

2. Use according to claim 1, characterized in that:

the tumor is breast cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, head and neck cancer, bladder cancer, cervical cancer, diffuse B large cell lymphoma, esophageal cancer, glioma, pancreatic cancer, prostate cancer, melanoma, thymoma or endometrial cancer.

3. Use according to claim 1 or 2, characterized in that:

the inhibitor is a substance for inhibiting the transcription or translation of CTD-2256P15.2 gene, a substance for promoting the degradation of CTD-2256P15.2 gene or the micro-peptide PACMP or a substance for inhibiting the biological function of the micro-peptide PACMP.

4. Use of the inhibitor and the other anti-tumor substance of any one of claims 1 to 3 in at least one of:

1) preparing a tumor treatment product;

the other anti-tumor substances are other anti-tumor drugs or reagents or instruments required by other anti-tumor treatment methods.

5. A product comprising the inhibitor of any one of claims 1-3, and, other oncology therapeutic agents or other agents or devices required for antineoplastic treatment;

the product has at least one of the following functions:

1) treating tumors;

3) improve the sensitivity of the tumor cells to the chemotherapeutic drugs.

The CTD-2256P15.2 or the encoded micro-peptide PACMP thereof can be used as the action target of tumor treating agents.

Application of CTD-2256P15.2 gene as marker in preparing products for evaluating sensitivity of tumor patients to chemotherapeutic drugs or predicting prognosis status of tumor patients.

8. The application of the substance for detecting the expression quantity of CTD-2256P15.2 in tumor tissues in the following steps:

9. The application of the substance and the data processing device for detecting the expression quantity of CTD-2256P15.2 in the tumor tissue is 1) to 2):

10. A system for predicting prognosis after chemotherapy of a patient with a tumor to be tested, comprising a substance for detecting the expression level of CTD-2256P15.2 in tumor tissue and the data processing device of claim 9.