CN114989286A

CN114989286A - Application of DDUP as tumor drug resistance detection, treatment and prognosis molecular target

Info

Publication number: CN114989286A
Application number: CN202210622773.8A
Authority: CN
Inventors: 李隽�; 宋立兵; 余汝媛; 李新城; 张淑霞; 胡雅梦
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2022-09-02
Also published as: WO2023231086A1

Abstract

The invention belongs to the field of biotechnology and medicine, and discloses application of DDUP as a molecular target spot for drug resistance detection, treatment and prognosis of tumors. The DDUP can regulate and control DNA damage and/or DNA damage repair, is obviously related to platinum drug resistance, relapse, overall survival time and disease-free survival time of tumor patients, and can be used as a marker for tumor drug resistance, tumor prognosis and tumor relapse detection.

Description

Application of DDUP as tumor drug resistance detection, treatment and prognosis molecule target spot

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to application of DDUP as a molecular target spot for drug resistance detection, treatment and prognosis of tumors.

Background

Malignant tumor seriously threatens human life health, and in 1943, scientists firstly apply nitrogen mustard to the treatment of lymphoma, and the sequence of modern tumor chemotherapy is uncovered. New antitumor drugs are continuously appearing, and chemotherapy becomes one of the main treatment methods of tumors, but as bacteria are easy to generate resistance to antibiotics, tumor cells also generate resistance to chemotherapeutic drugs, which is one of the main reasons for the failure of chemotherapy. Therefore, tumor drug resistance is one of the key problems to be solved urgently in the treatment of malignant tumors. Taking ovarian cancer, which is one of the malignant tumors of the reproductive system of three women, as an example, the ovarian cancer has serious threat to the health of women in the world due to high malignancy degree, high recurrence rate and high treatment difficulty. Because specific clinical symptoms are lacked in the early stage and no effective early screening and diagnosis method exists, the ovarian cancer is difficult to screen, and more than 70 percent of patients are diagnosed in the advanced stage of the ovarian cancer. At present, the standard treatment scheme after the ovarian cancer cytoreduction is a platinum-based chemotherapy scheme, and most patients with ovarian cancer can obtain clinical remission after initial treatment, but 70 percent of patients relapse after initial treatment, and the 5-year survival rate is only 39 percent. One of the fundamental reasons is platinum resistance, which leads to a gradual decrease in the reactivity of cancer cells to platinum during the course of treatment, ultimately leading to treatment failure. Therefore, the research on the specific mechanism of platinum resistance of ovarian cancer finds out the key factors for overcoming the platinum resistance, and is particularly important for improving the treatment effect of ovarian cancer.

The formation of tumor drug resistance is a biological process in which multiple genes and multiple factors participate in regulation, the formation mechanism is complex, and the research is not thorough at present. The main target of action of platinum drugs is DNA, and the platinum-DNA adduct is formed by forming a single adduct or intrachain crosslinking with DNA, so that DNA replication and transcription are inhibited, DNA breakage and error coding are caused, and the caused DNA damage can activate an apoptosis pathway by activating various signal transduction pathways, and finally cell death is caused. When the signal transduction pathway is abnormally activated or inhibited, the increase of the outflow or the reduction of the absorption of platinum leads to the reduction of the accumulation of platinum in cells, the abnormal activation of DNA damage repair pathways and the like, leads to the resistance and the apoptosis inhibition of cells to platinum drugs, and promotes the generation of tumor platinum chemotherapy resistance.

To protect DNA from damage induced by endogenous or exogenous toxic substances, mammalian cells have evolved a complex signaling pathway and repair network to remove or tolerate DNA damage, including the DNA Damage Response (DDR) pathway and DNA repair proteins. Platinum-based chemotherapeutic drugs secondarily induce DNA double strand breaks by covalently binding guanine at the N7 site of DNA to form adducts with DNA. Following platinum-based drug therapy, cancer cells undergo DNA damage repair or apoptosis, which determines whether they survive or die. DDR plays a key role in maintaining genomic integrity and the development of resistance. Genomic instability was found to be a hallmark of cancer, and DDR defects lead to increased risk of tumors. DNA repair pathways mainly include: nucleotide Excision Repair (NER), mismatch repair (MMR), Base Excision Repair (BER), non-homologous recombination repair (NHEJ), homologous recombination repair (HR), post-replication repair (PRR), and the like. The interaction and coordination between the different repair pathways leads to DNA damage repair and cell survival, and alterations in these pathways (aberrant activation or silencing) contribute to the development of sensitivity or resistance to platinum-based drugs. Failure to properly detect, repair, or respond correctly to DNA damage results in the development of a chemotherapy-resistant phenotype. Therefore, the further and deep research on the chemotherapy drug resistance mechanism of tumor cells provides more effective action targets for the treatment of tumors, and becomes a problem to be solved urgently.

Disclosure of Invention

The first aspect of the present invention is directed to providing a DDUP.

It is an object of the second aspect of the present invention to provide a biomaterial related to DDUP of the first aspect of the present invention.

The object of the third aspect of the present invention is to provide the use of the DDUP of the first aspect of the present invention and/or the biomaterial of the second aspect.

The fourth aspect of the invention aims at providing the application of the substance for detecting DDUP in preparing tumor drug resistance detection products, tumor prognosis detection products or tumor recurrence prediction detection.

The fifth aspect of the invention aims to provide the application of the ATR inhibitor in the preparation of the DDUP inhibitor.

The sixth aspect of the invention aims at providing the application of the DDUP inhibitor in preparing products for improving the sensitivity of tumors to platinum drugs.

It is an object of the seventh aspect of the present invention to provide a medicament.

An eighth aspect of the present invention is to provide a kit.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a DDUP, the amino acid sequence of which comprises:

a) MWLVECTGRDLTGLSCLLSMDRQPRRRQHVAGCRDVPPPLPQGSWGQTSPRHSILCSKSGCDLLGGGEYNGETSGEEFLAPAWTCRAQQAATWLSVQQTSHKALGPAGGAAMSSKLSPEEQFLSRIHFLRTFMCSVAGAELPGIPQATENGEGCRPARDPASSPSSLSMASVYTQCSSAQLVSALS (SEQ ID NO. 2); or

b) A sequence having 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No. 2.

In a second aspect of the present invention, there is provided a biomaterial related to DDUP of the first aspect of the present invention, the biomaterial comprising any one of a1) to a 4):

a1) a nucleic acid molecule encoding a DDUP of the first aspect of the invention;

a2) an expression cassette comprising the nucleic acid molecule of a 1);

a3) a vector comprising the nucleic acid molecule of a 1);

a4) a cell line comprising the nucleic acid molecule of a 1).

Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

Preferably, the cell line does not comprise propagation material.

In a third aspect of the present invention, there is provided the use of the DDUP of the first aspect and/or the biomaterial of the second aspect for the modulation of DNA damage and/or DNA damage repair.

Preferably, the use is for in vitro non-therapeutic purposes.

In a fourth aspect of the present invention, there is provided use of a substance for detecting DDUP in preparation of a tumor drug resistance detection product, a tumor prognosis detection product or a detection for predicting tumor recurrence.

Preferably, the substance for detecting DDUP comprises a substance for quantitatively detecting DDUP.

Preferably, the substance for detecting DDUP comprises a substance for detecting DDUP at a gene level and/or a protein level.

Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry (e.g., immunofluorescence analysis, reverse enzyme-linked immunosorbent assay, immunogold immunoassay), Western blotting, Northern blotting, PCR, and biochip.

Preferably, the immunohistochemistry is selected from the group consisting of: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay and immunocolloidal gold method.

Preferably, the substance for detecting DDUP is selected from: substances specific to DDUP, such as antibodies (preferably monoclonal antibodies) thereto; DDUP specific probes, gene chips, PCR primers and the like.

Preferably, the substance for detecting DDUP is selected from the group consisting of: DDUP antibody, DDUP specific probe, gene chip and PCR primer.

Preferably, the amino acid sequence of DDUP comprises:

Preferably, the nucleotide sequence of DDUP is shown as SEQ ID NO. 1.

Preferably, the product is a kit.

Preferably, the tumor comprises at least one of ovarian cancer, breast cancer, liver cancer, lung cancer, and gastric cancer.

Preferably, the tumor resistance drug comprises a platinum-based drug; further preferably, the tumor-resistant drug comprises at least one of carboplatin and cisplatin.

In a fifth aspect of the invention, there is provided the use of an ATR inhibitor in the preparation of a DDUP inhibitor.

Preferably, the ATR inhibitor comprises Berzosertib.

Preferably, the DDUP inhibitor comprises a substance that inhibits DDUP activity; further preferably, the DDUP inhibitor is a substance that inhibits DDUP phosphorylation.

Preferably, the substance that inhibits DDUP phosphorylation inhibits DDUP T174 site phosphorylation.

Preferably, the amino acid sequence of DDUP comprises:

In a sixth aspect, the invention provides an application of a DDUP inhibitor in preparing a product for improving the sensitivity of tumors to platinum drugs.

Preferably, the DDUP inhibitor comprises at least one of a substance inhibiting DDUP activity, a substance degrading DDUP, a substance reducing DDUP expression level; further comprises at least one of a substance inhibiting the activity of DDUP and a substance reducing the expression level of DDUP.

Preferably, the substance inhibiting DDUP activity comprises a substance inhibiting DDUP phosphorylation; further preferably, the substance inhibiting DDUP activity comprises an ATR inhibitor; still further preferably, the substance inhibiting DDUP activity comprises Berzosertib.

Preferably, the substance for reducing the expression level of DDUP comprises at least one of b1) to b 3):

b1) siRNA, dsRNA, miRNA, ribozyme, gRNA, or shRNA targeting DDUP;

b2) a nucleic acid molecule encoding b 1);

b3) an expression cassette, vector or transgenic cell line comprising b 2).

Preferably, the sequence of the shRNA targeting DDUP is shown as SEQ ID NO.3 or SEQ ID NO. 4.

Preferably, the sequence of the gRNA targeting DDUP is shown as SEQ ID NO.5 or SEQ ID NO. 6.

Preferably, the sequence of the siRNA targeting DDUP is shown as SEQ ID NO.21 or SEQ ID NO. 22.

Preferably, the DDUP inhibitor comprises at least one of c1) -c 10):

c1)Berzosertib；

c2) shRNA targeting DDUP;

c3) a nucleic acid molecule encoding c 2);

c4) an expression cassette, vector or transgenic cell line comprising c 3);

c5) shRNA targeting DDUP;

c6) a nucleic acid molecule encoding c 5);

c7) an expression cassette, vector or transgenic cell line comprising c 6);

c8) siRNA targeting DDUP;

c9) a nucleic acid molecule encoding c 8);

c10) an expression cassette, vector or transgenic cell line comprising c 9).

Preferably, the amino acid sequence of DDUP comprises:

Preferably, the nucleotide sequence of DDUP is shown as SEQ ID NO. 1.

Preferably, the platinum drug comprises at least one of carboplatin and cisplatin.

Preferably, the product is a medicament.

In a seventh aspect of the invention, there is provided a medicament comprising: DDUP inhibitors.

Preferably, the substance comprises at least one of a substance inhibiting DDUP activity, a substance degrading DDUP, and a substance reducing DDUP expression level.

Preferably, the DDUP inhibitor comprises at least one of a substance degrading DDUP, a substance reducing the expression level of DDUP; or at least one of a substance degrading DDUP, a substance reducing the expression level of DDUP and a substance inhibiting DDUP activity.

b1) siRNA, dsRNA, miRNA, ribozyme, gRNA, or shRNA targeting DDUP;

b2) a nucleic acid molecule encoding b 1);

b3) an expression cassette, vector or transgenic cell line comprising b 2).

Preferably, the sequence of the siRNA targeting DDUP is shown in SEQ ID NO.21 or SEQ ID NO. 22.

Preferably, the DDUP inhibitor comprises:

c2) -c 10); or

c2) C10) and c 1;

c1)Berzosertib；

c2) shRNA targeting DDUP;

c3) a nucleic acid molecule encoding c 2);

c4) an expression cassette, vector or transgenic cell line comprising c 3);

c5) shRNA targeting DDUP;

c6) a nucleic acid molecule encoding c 5);

c7) an expression cassette, vector or transgenic cell line comprising c 6);

c8) siRNA targeting DDUP;

c9) a nucleic acid molecule encoding c 8);

c10) an expression cassette, vector or transgenic cell line comprising c 9).

Preferably, the amino acid sequence of DDUP comprises:

Preferably, the nucleotide sequence of DDUP is shown as SEQ ID NO. 1.

Preferably, the medicament is for increasing the sensitivity of a tumor to a platinum-based drug.

Preferably, the medicament further comprises a platinum-based drug.

Preferably, the medicament further comprises at least one of cisplatin and carboplatin.

Preferably, the medicament is for the treatment of a tumour.

In an eighth aspect of the present invention, there is provided a kit comprising: detecting the substance of DDUP.

Preferably, the substance for detecting DDUP is selected from: DDUP antibody, DDUP specific probe, gene chip and PCR primer.

Preferably, the amino acid sequence of DDUP comprises:

Preferably, the nucleotide sequence of DDUP is shown as SEQ ID NO. 1.

Preferably, the kit is used for any one of d1) -d 3):

d1) detecting drug resistance of the tumor;

d2) detecting the prognosis of the tumor;

d3) detection of predictive tumor recurrence.

The invention has the beneficial effects that:

the invention discloses a DDUP for the first time; the DDUP can regulate and control DNA damage and/or DNA damage repair, is obviously related to platinum drug resistance, relapse, overall survival time and disease-free survival time of tumor patients, and can be used as a marker for tumor drug resistance, tumor prognosis and tumor relapse detection.

The invention discloses the application of a DDUP inhibitor in improving the sensitivity of tumors to platinum drugs for the first time, and through the combination of the DDUP inhibitor and the platinum drugs, the gamma-H2 AX of tumor cells can be obviously increased, the cell proliferation index ki67 is obviously reduced, the apoptosis proportion is obviously increased, the weight and the volume of the tumors are obviously inhibited, the survival period of patients is prolonged, and the tumor treatment effect is improved.

Drawings

FIG. 1 is a graph showing the effect of the micro-protein-DDUP encoded by LncRNA CTBP1-DT on DNA damage repair: wherein A is a result diagram of quantitative proteomics combined with ribosome profile analysis to identify new factors involved in DNA damage repair; b is a mass spectrum of the micro-protein-DDUP encoded by the LncRNA CTBP 1-DT; c is a schematic diagram of the position of CTBP1-DT on the genome, the putative DDUP ORF located in the second exon, and the full-length DDUP for the synthesis of anti-DDUP polyclonal antibody; d is the comet assay result chart in example 1; e is the immunofluorescence analysis results plot in example 1, as shown: compared with Vector, p is less than 0.001.

FIG. 2 is a graph showing the effect of LncRNA CTBP1-DT on DNA damage repair: wherein A is a schematic diagram of DDUP mutant vector construction and an expression result diagram thereof under CPT treatment; b is a diagram of the results of immunofluorescence analysis of the expression of DDUP mutant vectors under CPT treatment; c is the comet assay result chart in example 2; d is a chart of immunofluorescence analysis results of DDUP mutant vector under CPT treatment for detecting gamma-H2 AX focus; e is the result of the immunoblotting experiment in example 2; denotes: p < 0.001 compared to Vector/Ctrl.

FIG. 3 is a graph of the effect of DDUP phosphorylation on DDUP-regulated DNA damage repair: wherein A is a result graph of quantitative proteomics experiment identification of protein interacting with DDUP; b is a graph of the results of Co-IP analysis of DDUP interaction with ATR, ATM, RAD18, γ -H2AX and RAD 51C; c is a molecular docking scheme for ATR and DDUP; d is a graph of the results of Co-IP analysis of the interaction of full-length or truncated DDUP with ATR; e is a graph of the results of the interaction of DDUP and its mutants with pTQ/SQ in a Co-IP assay; f is a plot of immunofluorescence analysis results of γ -H2AX foci in cells transfected with different mutants; denotes: compared with Vector, p is less than 0.001.

FIG. 4 is a graph of the effect of DDUP on the retention of RAD18 at the site of DNA damage: wherein A is a result map of the co-location of the DDUP focus with the RAD18, γ -H2AX and RAD51C focus; b is a graph of the results of Co-IP analysis of DDUP interaction with RAD18, γ -H2AX and RAD 51C; c is the three-dimensional structure diagram of the simulated DDUP/WT and DDUP/T174D; d is a result graph of the change of the RAD18 and RAD51C focuses along with time; e is the result of the homologous recombination repair assay in example 4; f is a result graph of regulation and control of DDUP imbalance on PCNA monoubiquitination analyzed by co-immunoprecipitation and immunoblotting experiments; denotes: p < 0.001 compared to Vector/Ctrl.

FIG. 5 is a graph of the effect of DDUP on cisplatin resistance of ovarian cancer cells in vitro: wherein A is a relation graph of CTBP1-DT and progression-free survival, survival after progression or recurrence-free survival of tumor patients; b is a graph of the results of the effect of DDUP on Platinum drug Resistance (Platinum Resistance) and Relapse (Relapse); c is a result plot of the effect of DDUP on overall and disease-free survival times; d is a graph of the results of immunoblot analysis of chromosome binding and total DDUP expression in patient-derived ovarian cancer cells (PDOVC); e is a graph of the results of a homologous recombination repair assay for patient-derived ovarian cancer cells (PDOVC); f is a graph of results of immunoblot assays analyzing chromosome binding and expression of total monoubiquitinated PCNA and DDUP in patient-derived ovarian cancer cells (PDOVC); denotes: compared with PDOVCs #3/PDOVCs #4 without Berzosertib, p is < 0.001.

FIG. 6 is a graph showing the effect of DDUP on cisplatin resistance in ovarian cancer cells in vivo: wherein A is a schematic diagram of the construction of a patient-derived ovarian cancer xenograft model; b is a representative plot of xenograft tumors following different treatments; c is a graph of the effect of different treatments on the weight of xenograft tumors; d is a plot of the effect of different treatments on the volume of xenograft tumors; e is the Kaplan-Meier survival curve for mice treated differently; f is immunohistochemical staining and TUNEL plots of tumors from differently treated mice; denotes: compared with Vehicle, p is less than 0.001.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.

Interpretation of terms:

the mass spectrum data refers to data such as peptide fragment information, secondary spectrogram and the like obtained by using a mass spectrometer.

Ribosome profile high throughput sequencing data: refers to data obtained by high throughput sequencing of isolated ribosome-mRNA complexes.

Camptothecin: a chemotherapy medicine for inducing DNA injury is provided.

The PDX model: is a new generation tumor model established by inoculating tumor tissues excised by a patient operation into an immunodeficiency mouse. The method preserves the genotype and phenotype diversity of the tumor tissue of the patient, and can truly reflect the characteristics of the original tumor.

Focus: english is Foci, the complex form of focus, which originally means the focus. Used in DNA damage repair means that various damage repair proteins are recruited to the site of DNA damage. When DNA is damaged, repair proteins are recruited to form focuses at the damaged sites, and then the antibodies of the corresponding proteins are used for carrying out cell immunofluorescence staining, and a plurality of small spots formed by DNA damage related proteins can be observed in cell nuclei under a fluorescence microscope. This method can be used to detect recruitment of damage repair-associated proteins to sites of DNA damage.

Berzosertib: inhibitors of ATR kinase.

Example 1 micro-protein-DDUP encoded by LncRNA CTBP1-DT promotes DNA Damage repair

(1) Mass spectrum identification and ribosome map high-throughput sequencing combined identification of DNA damage repair novel protein DDUP

Mass spectrometric identification combined ribosome analysis: DNA damage was induced in 293T cells by treating 293T cells with Camptothecin (CPT) for 1 hour. And (3) identifying by LC-MS/MS mass spectrum: total proteins of a control group (DMSO) and a CPT treatment group were extracted respectively, and subjected to enzymolysis, and then divided into 10 groups for mass spectrometry, each group having two biological replicates. High throughput sequencing using ribosomal mapping: and (3) extracting ribosome RNA of the control group and the CPT treatment group respectively, establishing a library, and then carrying out high-throughput sequencing. The protein DDUP translated by lncRNA CTBP1-DT is identified by combining the mass spectrometry analysis and the ribosome map high-throughput sequencing result, and is related to DNA damage repair. The method comprises the following specific steps:

1) ribosome profile high-throughput sequencing:

firstly, cell plating and drug treatment: when the growth density of the cells reaches 80-90%, the cells are treated at a rate of 3.5 × 10 ⁶ The density of/mL was seeded on 10-cm dishes. The next day, the culture supernatant was discarded, and 10mL of a medium (CHX complete medium) (i.e., Cycloheximide, #5087390001, Sigma-Aldrich, St.Loui) containing the corresponding drug was addeds, Missouri, 100. mu.g/mL), 5% FBS in DMEM medium). Cell samples were collected after 1 hour.

Collecting cell samples: the cells were rinsed with pre-warmed PBS, replaced with CHX complete medium (i.e., Cycloheximide, #5087390001, Sigma-Aldrich, St. Louis, Missouri, 100. mu.g/mL, DMEM medium containing Cycloheximide, 5% FBS, incubated in a cell incubator at 37 ℃ for 3-4 minutes; the medium was discarded and washed once with 5mL of pre-cooled PBS; 300. mu.L of pre-cooled lysine buffer, lysine buffer:140mM NaCl, 5mM MgCl. sub.5 mM, was added to each dish) ₂ 10mM Tris-HCl pH 8.0, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.4U/. mu.L RNase inhibitor, 20mM DTT, 0.1mg/mL cycloheximide, 10mM RVC (vanadyl riboside complex), 0.1% (v/v) cocktail), and gently scraping the cell mixture with cell scraper and collecting into 1.5mL EP tubes. Repeatedly blowing and beating the cell suspension by using a gun, incubating for 15min, and inverting the EP tube for several times; centrifuge at 16,000 Xg for 10min at 4 ℃. Taking the supernatant for later use; the above operations were performed on ice.

Preparing a sucrose density gradient: preparing 10% (w/v) and 50% (w/v) sucrose solutions in advance; 5.5mL of 10% sucrose solution is added into an ultracentrifuge tube, and 5.5mL of 50% sucrose solution is carefully added into the bottom of the centrifuge tube by using a long needle, so that the solutions are prevented from being mixed; a density gradient pump was used to make the sucrose density ladder.

Fourthly, ultracentrifugation and separation of samples: gently adding the treated sample into a sucrose density gradient ultracentrifuge tube, ultracentrifuging with SW40Ti ultracentrifuge rotor at 170,000 Xg, and centrifuging at 4 ℃ for 2 h; and (3) separating and collecting the ultracentrifuged sample components in sequence by using a gradient component separation instrument, collecting the sample of each component after 80 seconds, and simultaneously obtaining the peak images of each component flowing out in sequence.

Fifthly, extracting and identifying component sample RNA: and (3) respectively adding the collected sucrose gradient samples into equal volume of Trizol solution, and extracting RNA according to a Trizol method.

Sixthly, library construction and quality inspection: using Ribo-Zero ^TM kit for removing rRNA from total RNA sample (part of lncRNA has polyA tail structure identical to mRNA, and rRNA removal method can be used for removing rRNA maximallylncRNA containing polyA tail was retained to some extent). Fragmentation buffer was added to the enriched RNA to break the RNA into small fragments. Then using the fragmented RNA as a template, adding 6bp random primers (random hexamers) to perform reverse transcription to synthesize a first cDNA chain, and adding buffer solution, dNTPs (dTTP in dNTP is replaced by dUTP), DNA polymerase I and RNase H to synthesize a second cDNA chain. After the synthesized double-stranded cDNA is purified, end repaired, A is added and a sequencing joint is connected for treatment, a U-containing cDNA second strand is degraded by USER enzyme and PCR enrichment is carried out, and finally, the PCR product is purified by AMPure XP beads to obtain a final strand specific library. After the library is constructed, firstly, carrying out preliminary quantification by using the Qubit 2.0, diluting the library, then, detecting the size of an insert of the library by using Agilent 2100, and after the insert accords with the expectation, accurately quantifying the effective concentration of the library by using a Q-PCR method to ensure the quality of the library.

Seventhly, machine sequencing: sequencing was performed using the Illumina high throughput sequencing platform NovaSeq HiSeq X Ten.

2) Mass spectrum identification:

firstly, cell plating and drug treatment: when the growth density of the cells reaches 80-90%, the cells are treated at a rate of 3.5 × 10 ⁶ The density of/mL was seeded on 10-cm dishes. The next day, the culture supernatant was discarded, and 10mL of medium (CPT, 10. mu.M) containing the corresponding drug was added. Cell samples were collected after 30 minutes.

Collecting cell samples: the medium was discarded, and the cells were rinsed 2 times with 1 × PBS buffer. Cells were harvested using cell scraping.

③ transfer the sample to a corresponding 10kD ultrafiltration tube and centrifuge at 12,000g for 20 min.

mu.L of Buffer1(8M urea/100mM Tris-HCl, pH 8.5) was added to each ultrafiltration tube to dissolve the denatured protein sufficiently.

Adding 20 μ L DTT (dithiothreitol, 100mM) solution, reacting at 37 deg.C for 2h to reduce disulfide bond.

Sixthly, 20 mu L of IAA (indole-3-acetic acid, 500mM) solution is added for reaction for 15min at room temperature in a dark place.

Seventhly, centrifuging 12,000g of the protein solution after reduction alkylation for 20min, and discarding the solution at the bottom of the collecting pipe. Add 200uL Buffer1 and centrifuge, repeat 2 times.

Add 200. mu.L Buffer2(8M urea/100mM Tris-HCl, pH 8.0), centrifuge at 12,000g for 20min, discard the bottom solution of the collection tube, repeat 1 time.

Ninthly, adding 200 mu L ammonium bicarbonate solution (25mM), centrifuging for 20min at 12,000g, discarding the solution at the bottom of the collection tube, and repeating for 2 times.

The collection tube is replaced with a new tube, 100 μ L pancreatin solution (0.01ug/uL) is added, and the reaction is carried out at 37 ℃ for 14 h.

Taking out the ultrafiltration tube, centrifuging for 20min at 12,000g, and collecting peptide fragments after enzymolysis.

Adding 100 μ L ammonium bicarbonate solution (25mM), centrifuging at 12,000g for 10min, collecting the bottom solution, and mixing with the filtrate

The solutions of (4) are combined; centrifuging, and freeze drying.

The freeze-dried samples were dissolved with 100. mu.L of mobile phase A (10mM ammonium formate, 5% (v/v) acetonitrile in water, pH 10.0); peptide fragment separation was performed on Agilent 1100HPLC with detection wavelength: ultraviolet 210nm, flow rate: 0.25 mL/min; the separation gradient was a linear gradient from 5-38% (v/v) of mobile phase B (10mM ammonium formate, 90% (v/v) acetonitrile in water, pH 10.0) over 60 minutes. Collect 1 tube every 1 minute in the gradient range, collect 15 tubes of elution solution altogether, centrifuge and dry for LC-MS analysis.

LC-MS/MS analysis: identification was performed using a Thermo Scientific Q exact mass spectrometer.

The results are shown in FIG. 1 at A, B: identifying the long-chain non-coding RNA-CTBP1-DT (with the sequence being ATGTGGTTGGTGGAGTGCACAGGCAGGGACCTCACTGGACTTTCCTGTCTGCTCAGCATGGACAGGCAGCCCAGGAGAAGGCAGCACGTGGCCGGGTGCAGGGACGTACCACCCCCACTTCCCCAGGGGAGCTGGGGTCAGACGAGTCCCAGGCACTCCATCCTCTGCAGCAAGTCAGGTTGTGATTTACTAGGGGGTGGTGAATATAATGGAGAGACTTCTGGGGAGGAATTCCTGGCTCCCGCGTGGACTTGCAGAGCTCAACAGGCAGCCACGTGGCTGAGTGTCCAGCAAACATCACATAAGGCTTTGGGTCCTGCAGGTGGGGCTGCCATGAGCAGCAAGCTCAGTCCAGAAGAACAGTTCCTCTCCAGGATCCACTTCCTGCGCACTTTTATGTGCAGTGTAGCTGGAGCAGAGCTCCCCGGAATTCCACAGGCAACTGAGAACGGAGAGGGATGCAGGCCAGCCAGGGATCCAGCGTCTTCCCCATCGTCACTCTCCATGGCCTCCGTCTACACACAGTGTTCGTCTGCACAGCTTGTCAGCGCGTTATCATGA, SEQ ID NO.1) with the highest upregulation in DNA damage through the integrated analysis of mass spectrum data and ribosome map sequencing data; after CPT (10 μ M) treatment, γ -H2AX was produced, indicating that cells were damaged after CPT treatment; the analysis result of LC-MS/MS shows that compared with the control group, the CPT treatment group identifies a new Protein translated by lncRNA CTBP1-DT, which indicates that the lncRNA CTBP1-DT can translate out the new Protein to adapt to the stress in response to the stress of DNA Damage, and the new Protein is named as DNA Damage regulated Protein, namely DDUP (the amino acid sequence is shown as SEQ ID NO. 2).

(2) Preparation of DDUP polyclonal antibody (as shown in C in figure 1): first, DDUP 1-83 and 94-179 amino acid sequences were synthesized as antigens. Two experimental-grade Japanese big ear rabbits were immunized with the prepared DDUP antigen, each rabbit being immunized with 0.25mg of antigen. The antigen was injected in four divided injections over the next 120 days, i.e. 0.26mg,0.27mg,0.28mg,0.29mg antigen per rabbit on days 14, 28, 42 and 95, respectively. On day 14 after the last antigen injection, two rabbits were each bled. The titer of the antibody was detected by ELISA assay. Affinity purification was then performed by attaching DDUP to a Sulfolink column.

(3) Comet assay:

firstly, one day ahead, 293T and HeLa cells are respectively treated in 3-4 multiplied by 10 in advance ⁵ the/mL was uniformly plated in six-well plates. The following day lipofection:

the solution A is: 1. mu.g of DDUP-psin-EF2 plasmid (available from Shandong Weizhen Biotech Co., Ltd.), 2. mu. L, Opti-MEM 125. mu.L of P3000 reagent; or ShRNA5nM (final concentration), Opti-MEM 125. mu.L;

the solution B is: 125 μ L of Opti-MEM solution 5 μ L Lipofectamine was added ^TM 3000, mixing uniformly.

Slowly adding the solution B into the solution A (do not reverse the sequence), slowly mixing by a liquid transfer machine, standing at room temperature for 5min, and uniformly dripping the mixture into a cell culture medium to be transfected. After 48 hours, the medium was discarded, and complete medium containing CPT at a final concentration of 10. mu.M or VP-16 at a final concentration of 10. mu.M was added and incubated for 30 minutes. Wherein Vector is psin-EF2 empty, sh-V is empty Vector control.

Wherein the shRNA sequences of the silent DDUP are respectively as follows:

ShRNA#1:5’-ACCTCGGAATGATGCAGACTCCTATCTCAAGAGGATAGGAGTCTGCATCATTCCTT-3’(SEQ ID NO.3)；

ShRNA#2:5’-ACCTCGTTCGTCTGCACAGCTTGTCATCAAGAGTGACAAGCTGTGCAGACGAACTT-3’(SEQ ID NO.4)。

② experiments are carried out according to the specification of a comet assay kit (Trevigen, MD, USA). Briefly described as follows: the medium was discarded and the cells were digested with pancreatin. Resuspend cells with cold PBS to a cell concentration of 1 х 10 ⁵ Then mixed with the melted LMAGarose agarose gel at a ratio of 1:10 (v/v) and spread evenly on a preheated (37 ℃ C.) slide. After the slide was cooled, the slide was soaked in the pre-cooled lysis solution for 1 hour, then soaked in 50mL neutral electrophoresis for 30 minutes, followed by electrophoresis for 1 hour (21V, 4 ℃). The electrophoresis solution was then removed, and the sample was immersed in the DNA precipitation solution for 30 minutes, further immersed in 70% (v/v) ethanol for 30 minutes, and then subjected to SYBR ^TM Gold (1:10000) staining. This experiment was repeated 3 times in parallel.

Measuring comet images by using an Opencomet plug-in, and randomly selecting 100 cells in each sample to calculate Tail distance (Tail Moment).

The experimental results are as follows: comet assay is a technique to detect DNA damage at the single cell level. Since γ -H2AX is a marker of DNA damage, DNA damage can be assessed by expression of γ -H2 AX. If the DNA is damaged, the fragmented pieces enter the gel and, under the influence of the electric field, the DNA pieces migrate away from the site towards the anode, forming a tail. Comet assay results are shown in fig. 1 at D: under the condition of DNA damage, compared with a control, the DDUP is highly expressed, and the tailing of DNA is obviously reduced; while with knockdown of DDUP, tailing of DNA was significantly increased; the high expression DDUP is shown to inhibit DNA damage, and the DDUP is knocked down to promote DNA damage.

(4) Immunofluorescence analysis: first, cells were previously treated at 2X 10 ⁴ PermL was plated on a 24-well plate covered with Cover slip. 293T and HeLa cells were subjected to DDUP overexpression or knockdown, respectively (same method as in (3) comet assay), and a complete culture medium containing CPT at a final concentration of 10. mu.M was added thereto and incubated for 30 minutes. The CPT-treated cells were treated as follows:

wash with PBS 2 times.

② adding 4 percent paraformaldehyde, and fixing for 30 minutes at room temperature.

③ the fixative was aspirated and washed 3 times with PBS.

(iv) perforating the cells with 0.5% Triton X-100 solution and incubating the cells at room temperature for 10 minutes.

Using 1 XPBST on the shaker washing, repeated 3 times, each time for 5 minutes.

Sixthly, adding 1 percent BSA, and sealing for 45 minutes at room temperature.

Seventhly, cells are incubated overnight with primary antibody (1:800) prepared by gamma-H2 AX antibody.

Recovering primary antibody incubation solution, washing with 1 × PBST on a shaking table for 5min each time, and repeating for 3 times.

Ninthly, incubation of the second antibody: preparing a rabbit secondary antibody with the primary antibody corresponding to the resistance according to the ratio of 1:500, and adding the rabbit secondary antibody into a hole to incubate the cells for 1 hour at normal temperature and in dark.

The fractions were washed on a shaker with 1 XPBST 5min each time and repeated 3 times.

Nuclei were stained with DAPI solution at room temperature for 5 min. Wash with 1 XPBST on a shaker for 5min each time, repeat 3 times.

After air drying, the cells were mounted with anti-quenching mounting medium, imaged using confocal microscope Zeiss LSM880, and the number of foci formed per cell nucleus was counted and counted using ImageJ software.

The experimental results are as follows: since γ -H2AX is a marker of DNA damage, DNA damage can be assessed by expression of γ -H2 AX. The results of the immunofluorescence experiments are shown in fig. 1E: under the condition of DNA damage, compared with a control, the focus formed by high-expression DDUP and gamma-H2 AX is obviously reduced, and the focus formed by knocking down DDUP and gamma-H2 AX is obviously increased; the high expression DDUP is shown to inhibit DNA damage, and the DDUP is knocked down to promote DNA damage.

Example 2 LncRNA CTBP1-DT does not affect DNA damage repair

(1) Construction of DDUP mutant vector (a in fig. 2): the vector is constructed in pSin-EF1a-puro-oligo vector (Addge #85132) by using DDUP, 5 'UTR-DDUP-3' UTR, 5 'UTR-ATG 1(m) -3' UTR, 5 'UTR-ATG 2(m) -3' UTR, 5 'UTR-ATG 1/2(m) -3' UTR sequences (ATG1(m), ATG2(m) respectively refer to the mutation of the first and second ATGs in SEQ ID NO.1 into ATT) through restriction sites of BamH1 and EcoR1, and the DDUP ORF frame of each vector is provided with a Flag tag.

The construction and identification method of the vector is as follows:

firstly, primer design: the plasmid construction primers involved in this study are shown in Table 1.

Amplification of target sequence by PCR

a. After extracting RNA from 293T cells using trizol (life technologies) and reverse-transcribing the RNA into cDNA, a PCR amplification reaction system was prepared in a 0.2mL sterile EP tube using the cDNA as a template (# F1066K, TOYOBO): 293T cDNA 100. mu.g, dNTPs (2.5mM) 5. mu.L, 10 XBuffer 5. mu.L, DNA polymerase (KOD plus Neo) 1. mu.L, Primer-up (10. mu.M) 1.5. mu.L, Primer-dn (10. mu.M) 1.5. mu.L, and ultra pure water 35. mu.L.

b. And (3) sufficiently and uniformly mixing the reaction solution, then performing transient instantaneous centrifugation, and placing the mixture into a PCR instrument for PCR amplification, wherein the reaction conditions are as follows: 94 ℃ for 2 min; 10s at 98 ℃,30 s at 58 ℃, 1min at 68 ℃ and 30 cycles; 7min at 68 ℃;

c. preparing 1% agarose gel (adding ethidium bromide with final concentration of 0.5 mug/mL), loading the PCR amplification product, performing electrophoresis, cutting off corresponding bands of the agarose gel according to the size of the bands under the irradiation of an ultraviolet lamp, and recovering the gel and purifying to extract the amplification product.

PCR amplification of target sequence

Gel recovery and purification were performed by QIAquick Gel Extraction Kit, following the instructions in the Kit:

a. transferring the cut agarose gel to a sterile 1.5mL EP tube, chopping, and weighing the gel block;

b. adding Buffer QG with 3 times of gel volume according to 1 μ g/1 μ L, mixing thoroughly, heating in water bath at 50 deg.C for 10min until the gel block is completely melted, shaking every 5min, and mixing gel solution;

c. transferring the melted gel mixed solution to a Spin Column arranged on a collecting pipe, and marking;

d. putting the collecting pipe into a centrifuge at 12000rpm for 1min, and discarding the filtrate;

e, adding 750 mu l of BufferPE into spin Column, re-centrifuging at 12000rpm for 30s, and discarding the filtrate;

f. putting the collecting pipe into a centrifuge for hollow separation at 12000rpm for 2 min;

g. spin Column was mounted on a new 1.5ml sterile EP tube and labeled. After preheating the Elution Buffer at 65 ℃, vertically adding 50 μ l of Elution Buffer at the center of the Spin Column membrane, and standing for 1min at room temperature;

h. centrifuging at 12000rpm for 1min in a centrifuge, and eluting to obtain the collected DNA solution.

Vector plasmid and PCR product double enzyme digestion

a. The cleavage reaction of the plasmid vector with the PCR product was as follows ((New England Biolabs, MA, USA)): Psin-EF1a-puro 1. mu.L, BamH1(# R0136S) 1. mu.L, EcoR1(# R0101S) 1. mu.L, 100 XBA 0.5. mu.L, 10 XBuffer 5. mu.L, enzyme-free water 41.5. mu.L. PCR product 42.5. mu.L, BamH1(# R0136S) 1. mu.L, EcoR1(# R0101S) 1. mu.L, 100 XBA 0.5. mu.L, 10 XBuffer 5. mu.L.

b. Mixing, centrifuging for 2-3h at 37 deg.C, electrophoresing the product with 1-2% agarose gel (containing ethidium bromide with final concentration of 0.5 μ g/ml), cutting the agarose gel according to the display position of target band under ultraviolet lamp, and recovering and purifying.

Fifth connecting the target sequence with the vector plasmid

a. The ligation reaction was configured as follows (New England Biolabs, MA, USA): mu.L of the target sequence, 3. mu.L of Psin-EF1a-puro 1. mu.L, 1. mu.L of 10 XLigase Buffer, 0.5. mu.L of T4 DNA Ligase (# M0202S), and 4.5. mu.L of ultrapure water.

b. And lightly blowing and beating to fully and uniformly mix the mixed solution, and connecting for 2-3h at room temperature.

Conversion of product

a. Taking out 100 μ L of competent bacteria (# CB101, TIANGEN) from-80 deg.C, melting on ice, and gently blowing and stirring thoroughly;

b. slowly adding 10 μ L of the ligation product into the competent bacteria solution, gently mixing, and standing on ice for 30 min;

c. putting the mixed solution of the b into a water bath kettle at 42 ℃, immediately taking out the mixed solution after the heat shock reaction is carried out for 90s, and putting the mixed solution on ice for 2-3 min;

d. to the mixture of c was added 900. mu.L of LB medium (10 g of peptone (Tryptone), 5g of Yeast Extract (Yeast Extract) and 10g of NaCl powder, respectively, dissolved and made to 1L of ddH ₂ Sterilizing in O (distilled water) at high temperature and high pressure for later use), and placing into shaking table to shake bacteria for 1h at 230rpm and 37 ℃;

e. dripping 400 mu L of the mixed solution of the transformed bacteria liquid on a culture dish containing a solid culture medium (weighing 15g of Agarose powder, adding the Agarose powder into 1L of LB culture medium, dissolving, then sterilizing at high temperature and high pressure, pouring into a culture dish of 100mm, cooling to obtain the solid culture medium), inclining the culture dish to enable the bacteria liquid to be uniformly distributed, and standing for 10min at room temperature;

f. and then, inversely placing the culture dish in a constant-temperature incubator at 37 ℃ for culture, and carrying out bacterium picking and identification after independent bacteria visible to the naked eye grow out for 12-16 hours.

Seventhly, extracting recombinant plasmid DNA

Colonies with independent growth and moderate size were selected from the petri dish, transferred to 2mL of LB broth (containing ampicillin), placed on a shaker, shaken at 230rpm overnight (14-16 h), 37 ℃. Extraction of small amounts of plasmid was performed by QIAprep Spin miniprep kit, operating as described:

a, sucking 2mL of turbid bacterial liquid into a 2mL sterile EP tube, placing the sterile EP tube in a centrifuge, centrifuging at 12000rpm for 1min, and removing supernatant;

b. adding 250 mu L of Buffer P1 (containing RNase A1) into an EP tube, and blowing and beating to fully reselect and fully suspend the precipitated thalli;

c. adding 250 mu L of Buffer P2, slightly turning the EP tube up and down, mixing for 4-6 times until the bacterial liquid becomes transparent viscous solution, namely the bacterial liquid is completely cracked;

d. adding 400 mu l of Buffer N3 into an EP tube, slightly turning the EP tube up and down, mixing for 4-6 times until white flocculent precipitate appears in the mixed solution, namely the cracking is stopped, putting the EP tube into a centrifuge for centrifugation at 12000rpm for 10min at room temperature;

e. carefully aspirate the supernatant and transfer to a QIAprep Spin Column tube at 12000rpm, centrifuge for 1min and discard the filtrate;

f. adding 500 mu LBuffer PB into Spin Column at 12000rpm, centrifuging for 30s, and discarding the filtrate;

g. adding 750 mu L of Buffer PE into Spin Column, rotating at 12000rpm, centrifuging for 30s, and then discarding the filtrate;

h. placing the Column tube with the filtrate removed in a centrifuge, and separating for 2min at 12000 rpm;

i. the Spin Column was placed on a new sterile 1.5mL EP tube, the Elution Buffer was preheated in a 65 ℃ water bath, then 60. mu.L of Elution Buffer was carefully added to the center of the Spin Column membrane, left to stand at room temperature for 1min and centrifuged at 12000rpm for 1min, and the eluted solution was the DNA solution.

TABLE 1 plasmid construction primer sequences

According to the lipofection method in example 1, corresponding plasmids are transfected into 293T cells, after 48 hours, the culture medium is discarded, complete culture solution containing CPT with the final concentration of 10 mu M is added for incubation for thirty minutes, and then the proteins are extracted for carrying out the immunoblotting experiment, which comprises the following specific steps:

extracting protein: cells were rinsed 2 times with 1 × PBS buffer, and the rinse solution was aspirated off thoroughly for the last time. Appropriate amount of cell lysate (#89901, ThermoFisher Scientific) was added and lysed at room temperature for 5-10 min. The lysed cells were then disrupted sufficiently with a sonicator until the liquid was clear and flowing smoothly. Protein samples were quantitated according to the procedure of the BCA protein quantitation kit (#23225, ThermoFisher Scientific) instructions.

Preparing glue: a polypropylene gel was prepared at a concentration of 15.5%.

③ sample loading: samples are sequentially loaded according to experimental groups, and if the volume difference of two adjacent groups of samples exceeds 2 times, the samples are supplemented by 1 × Loading Buffer (# P0015L, Biyun).

And fourthly, electrophoresis: concentrating at constant pressure of 80V, separating at constant pressure of 120V, and stopping when bromophenol blue in the sample migrates to the bottom of the separation gel.

Turning the film: the Transfer clips were soaked in 1 XTRIFER Buffer (10 XTRIFER Buffer: 145g of Glycine powder and 29g of Trisbase powder were weighed out and dissolved in ddH ₂ And O, fully stirring, then using sterile water to fix the volume to 1000ml, and storing at room temperature. 100ml of 10 × Transfer Buffer before use, diluted with 900ml of sterile water to 1 × Transfer Buffer), thoroughly moistened, then placed filter paper, gel and PVDF membrane (#1620177, Bio-rad) in this order with the black side facing down, carefully aligned up and down, avoiding the formation of air bubbles, and finally covered with filter paper and clamped in a rotating membrane holder. And (3) placing the film transferring clamp into a film transferring groove according to the correct direction, quickly adding 1 × Transfer Buffer to a horizontal line, transferring the film transferring device into a refrigeration house, and transferring the film for 2h at a constant current of 260 mA.

Closing: and (3) quickly taking out the PVDF membrane after the membrane conversion is finished, immediately soaking the PVDF membrane into 5% skim milk, and sealing the PVDF membrane for 30min at room temperature.

And the first reaction: a Flag primary antibody (#14793, CST) diluent (1:1000) is prepared by using 5% skim milk, and a PVDF membrane and a primary antibody are simultaneously put into an antibody incubation bag, sealed after air bubbles are completely discharged through extrusion, and incubated overnight at 4 ℃ in a horizontal shaking table.

And (v) cleaning: recovering primary antibody diluent, soaking PVDF membrane in 1 × TBST, washing the redundant antibody in a vertical shaking table for 5min for 3 times.

Ninthly, secondary antibody: a dilution (1:2000) of a mouse secondary antibody (# PR30012, proteintech) is prepared by using 5% skimmed milk, a PVDF membrane and the secondary antibody are placed into an antibody incubation bag at the same time, air bubbles are discharged, then the bag is sealed, and the bag is placed into a horizontal shaking table for incubation for 45min at normal temperature.

Cleaning the red: recovering the secondary antibody diluent, soaking the PVDF membrane in 1 XTSST, placing in a vertical shaking table to wash the redundant antibody for 3 times, 5min each time.

And (3) developing: putting the cleaned PVDF film into a cassette in advance, and immediately covering a transparent plastic film to avoid dry films. In a dark room, equal volumes of chemiluminescence solution A and solution B (# WP2005, ThermoFisher Scientific) are taken, mixed uniformly and then dripped on the surface of a PVDF membrane immediately, and the cassette is rotated gently to enable the luminescence solution to cover the whole membrane fully. And standing for 30s, covering a plastic film again, absorbing redundant luminous liquid by using absorbent paper, quickly covering 3-4X-ray films for exposure, adjusting exposure time according to the strip brightness, and developing and washing.

The results of the experiment are shown as a in fig. 2: compared with the 5 'UTR-ATG 1(m) -3' UTR and 5 'UTR-ATG 1/2(m) -3' UTR groups, the DDUP, 5 'UTR-DDUP-3' UTR and 5 'UTR-ATG 2(m) -3' UTR experimental groups can translate DDUP protein under the action of a DNA damage inducer, and the DDUP ORF expression frame is fused with tag protein Flag, so that the detection can be carried out by using a Flag antibody. Indicating that the translation of DDUP depends on the first ATG and that the second ATG does not function.

(2) Immunofluorescence analysis of the expression of DDUP mutant vectors under CPT treatment: the experimental procedure was the same as in (4) immunofluorescence assay of example 1, except that a different plasmid (DDUP mutant vector in (1) of this example) was transfected and the primary antibody used was different (primary antibody of this example was anti-Flag antibody (#14793, CST)), and the experimental results are shown in FIG. 2B: compared with the 5 'UTR-ATG 1(m) -3' UTR and 5 'UTR-ATG 1/2(m) -3' UTR groups, the experimental group of DDUP, 5 'UTR-DDUP-3' UTR and 5 'UTR-ATG 2(m) -3' UTR can translate DDUP protein under the action of DNA damage inducer, and the DDUP ORF expression cassette fuses tag protein Flag, so that the detection can be carried out by using Flag antibody; it is shown that the translation of DDUP depends on the first ATG, while the second ATG does not function, and more importantly, DDUP can form a focus under the induction of DNA damage.

(3) Comet experiments: the experimental procedure was the same as in (3) comet assay of example 1, except that a different plasmid (DDUP mutant vector in example (1)) was transfected, and the experimental results are shown in fig. 2, C: DDUP, 5 'UTR-DDUP-3' UTR and 5 'UTR-ATG 2(m) -3' UTR experimental groups, under the action of a DNA damage inducer, because of large-scale expression of DDUP, DNA has almost no tailing, DNA damage is effectively repaired, and DNA damage is less; in contrast, in the groups of 5 'UTR-ATG 1(m) -3' UTR and 5 'UTR-ATG 1/2(m) -3' UTR, since DDUP protein could not be expressed, DNA tailing was long and DNA damage could not be repaired.

(4) Immunofluorescence assay of DDUP mutant vectors under CPT treatment to detect γ -H2AX foci: the experimental procedure was the same as in (4) immunofluorescence assay in example 1, except that a different plasmid (DDUP mutant vector in example (1)) was transfected, and the experimental results are shown in fig. 2, D: under the action of a DNA damage inducer, due to the large-scale expression of DDUP, a focus formed by gamma-H2 AX is hardly seen in an experimental group of DDUP, 5 'UTR-DDUP-3' UTR and 5 'UTR-ATG 2(m) -3' UTR, which indicates that DNA damage is effectively repaired; in contrast, in the 5 'UTR-ATG 1(m) -3' UTR, 5 'UTR-ATG 1/2(m) -3' UT group, since DDUP protein could not be expressed, focus of gamma-H2 AX formation could be seen in large amount, and DNA was significantly damaged.

(5) Immunoblotting experiments: the experimental procedure was the same as in (1) of this example, differing only in the use of a different cell line and the use of a recombinant protein of DDUP as an indicator, as follows: a DDUP knockout cell line was constructed in HeLa cells using CRISPR-Cas9 system: the Guangzhou GeneChem company is responsible for designing specific sgRNA of DDUP and constructing GV392 plasmid to provide corresponding virus liquid, HeLa cells infect Cas9 virus liquid according to 15MOI virus concentration, after infection for 24 hours, infected cells are screened for 7 days by 0.5 mu g/mL puromycin, and stable HeLa/Cas9 cells are screened for re-infection with virus liquid (15MOI) of GV392-GFP-TRIM37 gRNA. After > 95% of the cells successfully infected were guaranteed by the infection efficiency of GFP, the successfully infected cells were isolated into single cell clones using flow cytometry. DDUP gRNA #1 and gRNA #2 represent monoclonal 1 and monoclonal 2, respectively. After cellular DNA of HeLa/DDUP gRNA #1 and gRNA #2 is extracted by QIAGEN gene DNA kit, the target gene sequence of gRNA is amplified by PCR, the amplified product is digested and digested by T7 endogenous nuclease 1(T7E1), and then the knockout efficiency of the target gene is detected by pEASY-T1 cloning kit. The gRNA sequences used were: gRNA # 1: 5'-GGTTGGTGGAGTGCACAGGCAGG-3' (SEQ ID NO. 5); gRNA # 2: 5'-TGCACAGGCAGGGACCTCACTGG-3' (SEQ ID NO. 6). The results of the experiment are shown in fig. 2E: after complete knockout of DDUP in HeLa is carried out by using a CRISPR/Cas9 gene knockout system, an immunoblotting experiment shows that the DDUP protein expression induced by CPT is obviously limited; more importantly, the DNA damage induced by the CPT in the immunofluorescence experiment is more obvious and is shown as the obvious increase of the gamma-H2 AX focus (a control group is wild-type HeLa cells, r-DDUP is recombinant protein of DDUP (the full-length protein of the DDUP is used for preparing the recombinant protein), a DDUP expression frame sequence is synthesized in vitro and is constructed into a PETE-28a vector, the insertion site is (Nco1/Xho1), an expression strain is E.coli Rosetta and is customized by the martian Egtaike biotechnology limited).

Example 3 interaction of DDUP with DDR protein

(1) Quantitative proteomics experiments identified proteins interacting with DDUP (a in figure 3):

liposome transfection (DDUP-psin plasmid) and drug treatment were carried out according to the experimental method of comet assay (3) in example 1.

② the CPT treated cells were placed on ice. After washing the cells twice with 1 × PBS, PBS was completely removed.

③ Add 1mL cell lysate containing protease inhibitor Cocktail (# P8340, Sigma-Aldrich) per 100mm dish, scrape and collect cell lysate with cell spatula, collect it in 1.5mL centrifuge tube, insert it on ice, vortex and shake for 30s every 10min, total lysis time 30 min.

Fourthly, the lysate is centrifuged at 12000rpm and 4 ℃ for 15min, the supernatant is collected, and 30 mu L of the supernatant is reserved to be used as an Input control and kept at-40 ℃ for standby.

Fifth, the DDUP antibody is coupled to the beads (#20423, ThermoFisher Scientific) in advance, 40. mu.L of the antibody obtained in (2) in example 1 is taken into a new 1.5mL centrifuge tube, 1 XPBS 500. mu.L/tube is taken, and the beads are washed for 2-3 times by resuspension.

Sixthly, adding the pretreated beads into the hydrolysate, and incubating overnight at 4 ℃ by rotating a shaker.

Seventhly, the bead-protein mixture is washed by the washing solution in a way of being slightly reversed from top to bottom, and the washing is repeated for 5 to 6 times.

If mass spectrometry is subsequently performed, the bead-protein mixture is subjected to enzymatic hydrolysis and detection using a mass spectrometer according to the mass spectrometry identification method of example 1.

Ninthly, if the immunoblotting experiment is performed subsequently, the Input sample is taken out, 30. mu.L of cell lysate (#89901, ThermoFisher Scientific) is added, and 30. mu.L of cell lysate (#89901, ThermoFisher Scientific) is also added to the IP sample. Then 5 Xloading buffer (# P0015L, Byunnan) was added according to a ratio of 1:5, and heated in a metal bath at 100 ℃ for denaturation for 10 min. The samples were subsequently subjected to immunoblotting for experiments. The results are shown in FIG. 3 as A: after DNA damage, a total of 12 proteins were identified, of which 4 were known to be associated with DNA damage, including ATR, γ -H2AX, RAD18, RAD 51C.

(2) Co-IP analysis of DDUP interaction with ATR, ATM, RAD18, γ -H2AX and RAD 51C:

the experimental procedure was the same as in (1) in this example, except that: berzosertib (final concentration 80nM) inhibitor with or without ATR; DNA damage was induced by CPT or CDDP (final concentration 10. mu.M). DDUP is co-immunoprecipitated by co-immunoprecipitation, and further immunoblotting experiments are carried out on DDUP-bound proteins. The results are shown in FIG. 3 as B: in the case of DNA damage (either CPT or CDDP treatment), DDUP binds to ATR, γ -H2AX, RAD18, RAD51C, but not to ATM, indicating DNA damage, DDUP interacts with ATR, γ -H2AX, RAD18, RAD 51C. This result is consistent with the mass spectrometry result of a in fig. 3.

(3) Molecule docking:

obtaining a simulated DDUP three-dimensional structure by utilizing an I-TASSER (https:// zhangglab. ccmb. med. umich. edu/I-TASSER /) server; the three-dimensional structure of ATR (PDB ID:5yz0) was downloaded from the RCSB protein database; using cluspro (f)https://cluspro.org) The server performs molecular docking, the result of which is shown as C in fig. 3: the N-terminal of DDUP, i.e., the region 21-53 aa, was bound to ATR, and the Heat repeats region of ATR was bound to DDUP, further confirming the binding of DDUP to ATR from the structural simulation.

(4) Co-IP assay interaction of full-length or truncated DDUP with ATR:

the experimental procedure was the same as in (1) in this example, except that: the carrier containing DDUP and the truncation thereof is transfected by liposome (the DDUP truncation comprises Flag-DDUP/N (comprising 1-62 th amino acid of DDUP), Flag-DDUP/deltaC (comprising 1-124 th amino acid of DDUP) and Flag-DDUP/C (comprising 125-186 th amino acid of DDUP), and the construction method of the carrier is the same as that of (1) in the embodiment 2); co-immunoprecipitation was performed using ATR antibody-conjugated beads (#20423, ThermoFisher Scientific). The results are shown in fig. 3D: ATR binds to Flag-DDUP/N, Flag-DDUP/Δ C, but not Flag-DDUP/C, indicating that ATR binds to the N-terminus of DDUP, which is also consistent with the previous assay results.

(5) Co-IP analysis of interaction of DDUP and its mutants with pTQ/SQ

The experimental procedure was the same as in (1) in this example, except that: liposome transfection contains DDUP/WT (DDUP-psin vector in example 2) or DDUP mutant DDUP/T174A vector (DDUP/WT vector in example 2 is used to mutate T at position 174 into A by point mutation technology), and the vector construction method is the same as that in example 2 (1); berzosertib (final concentration 80nM) as an inhibitor with or without ATR; Gamma-PPase protein phosphatase (2U/. mu.g) was used or not. The results are shown in FIG. 3 as E: through analyzing the sequence of DDUP, finding ATR specific phosphorylation site T174, and finding that ATR can not phosphorylate DDUP under the condition of DNA damage after mutating T174 site; also, ATR cannot phosphorylate DDUP in the presence of the inhibitor of ATR, Berzosertib. The above results indicate that ATR specifically phosphorylates DDUP T174 site.

(6) Focus of γ -H2AX in cells transfected with different mutants:

the experimental procedure was the same as in (1) in this example, except that: different DDUP mutants (DDUP/WT and DDUP/T174A, DDUP/T174D) or empty Vector (Vector) were transfected in DDUP knock-out HeLa cells, with or without inhibitor of ATR-Berzosertib (final concentration 80 nM). The results are shown in fig. 3F and fig. 4C: in HeLa/DDUP ^-/- In cells, upon DNA damage, substantial formation of γ -H2AX foci was observed at the mutant T174A site; in contrast, little focus was observed at the mutant T174D site at γ -H2 AX; DDUP/T174D can simulate the phosphorylation state of DDUP by ATR, and the whole structure is in an open state, which is more favorable for the combination of DDUP and other proteins; in the stable cell strain of DDUP/WT, when DNA is damaged, Berzosertib can obviously induce gamma-H2 AX focus; it can be seen that DDUP promotes DNA damage repair and that the focus of reducing γ -H2AX is ATR dependent phosphorylation.

Example 4 DDUP enhances the retention of RAD18 at the site of DNA damage

(1) The DDUP focus is co-located with the RAD18, γ -H2AX, and RAD51C focus:

the experimental procedure was the same as in (4) immunofluorescence assay in example 1, except that: two different primary antibodies (DDUP/RAD18, DDUP/γ -H2AX, DDUP/RAD51C antibody combinations) were used for each experimental group. The results are shown as a in fig. 4: in case of DNA damage, the foci formed by DDUP can be co-localized with the foci formed by RAD18, γ -H2AX, RAD51C, respectively.

(2) Co-IP assay DDUP interacts with RAD18, γ -H2AX and RAD 51C:

the experimental procedure was the same as in (1) of example 3, except that: interfering RNA of DDUP, RAD18, h2a.x were lipofected with the sequences shown in table 2. The results are shown in fig. 4 as B (scr is control): when DDUP is knocked down, and proteins co-immunoprecipitated by using gamma-H2 AX are used for immunoblotting experiments in case of DNA damage, the result shows that gamma-H2 AX can be combined with RAD18 and RAD51C, which indicates that the combination of gamma-H2 AX with RAD18 and RAD51C is independent of DDUP; the RAD18 is knocked down, and the immunoblotting experiment is carried out on the protein obtained by the co-immunoprecipitation of DDUP when DNA is damaged, and the result shows that DDUP can only be combined with gamma-H2 AX, which indicates that DDUP is not directly combined with RAD 51C; and the result of immunoblotting experiments on proteins co-immunoprecipitated by RAD18 with the reduced RAD18 shows that RAD18 can be combined with DDUP and RAD51C in the case of DNA damage, which indicates that the combination of RAD18 with DDUP and RAD51C is independent of gamma-H2 AX. Taken together, the above results indicate that DDUP binds directly to γ -H2AX, RAD18 to form a complex.

TABLE 2 sequences of interfering RNAs

Si RAD18#1	5’-GACCAAAGAGACACGTTCTGT-3’(SEQ ID NO.19)
		Si RAD18#2	5’-GCTGTTTATCACGCGAAGAGA-3’(SEQ ID NO.20)
Si DDUP#1	5’-GGAAUGAUGCAGACUCCUAUC-3’(SEQ ID NO.21)
		Si DDUP#2	5’-GTTCGTCTGCACAGCTTGTCA-3’(SEQ ID NO.22)
Si H2AX#1	5’-TGGACTAATTTTATTAAAGGATT-3’(SEQ ID NO.23)
		Si H2AX#2	5’-GACTAATTTTATTAAAGGATTGT-3’(SEQ ID NO.24)

(3) Simulated DDUP phosphorylation:

and obtaining the simulated DDUP/WT three-dimensional structure by utilizing an I-TASSER (https:// zhangglab. ccmb. med. umich. edu/I-TASSER /) server. The T174 site of the DDUP is mutated into D174, and an I-TASSER (https:// zhangglab. ccmb. med. umich. edu/I-TASSER /) server is utilized to obtain a simulated DDUP/T174D three-dimensional structure. The results are shown in fig. 4 as C: DDUP/T174D can simulate the phosphorylation state of DDUP by ATR, and the whole structure is looser, thus being more beneficial to the combination of DDUP and other proteins.

(4) Immunofluorescence assay RAD18 and RAD51C foci were analyzed over time:

the experimental procedure was the same as in (4) in example 1 except that: in DDUP ^-/- In the knockout cells, DNA damage is induced, and cells are harvested at 0, 0.5, 6 and 12 hours for immunofluorescence experiments. 100 cells were counted randomly per group, and cells with greater than 10 foci per nucleus were positive cells. The results are shown in fig. 4D: in control cells, when no DNA damage occurred (i.e., 0 hours), the immunofluorescence results indicated that RAD18 and RAD51 were evenly distributed in the nucleus, with little focus; after 10 minutes of DNA damage, RAD18 and RAD51 foci rapidly increased to 30 minutes, reaching plateau; then as time progressed, the RAD18 and RAD51 focus slowly decreased, by 24 hours, returning to background values; in contrast, in DDUP ^-/- In cells, due to complete knockout of DDUP, after DNA damage reaches a plateau, RAD18 and RAD51 foci rapidly decrease, returning to background by 12 hours; the above results indicate that DDUP can maintain the focus of RAD18 and RAD51 at the damaged chromosomal site for a long period of time to promote DNA repair.

(5) Homologous recombination repair detection experiments:

firstly, HeLa/DDUP are added ^-/- -#1、HeLa/DDUP ^-/- - #2 cells were treated at 3X 10 cells, respectively ⁵ The density per mL was uniformly spread in a six-well plate.

Secondly, after the cells are cultured to about 70 percent of the density, the empty load and DDUP plasmid (DDUP-psin plasmid in (3) of example 1) is transfected into HeLa cells according to the liposome transfection method; DDUP/WT, DDUP/T174A plasmid (DDUP/T174A in (5) of example 3)Vectors) were transfected into DDUP separately ^-/- -#1、DDUP ^-/- - #2 cells.

③ 24 hours later, the medium was replaced, and the DR-GFP plasmid (Addgene #26475) and pCBASCEI (Addgene #26477) plasmids were co-transfected into HeLa, HeLa/DDUP, respectively ^-/- -#1、HeLa/DDUP ^-/- - #2 cells.

Fourthly, after 48 hours, digesting by using trypsin and collecting cells; cells were washed with 1 × PBS, centrifuged at 1000rpm, 5min, and repeated twice.

Resuspend the cells with 200. mu.L of 1 XPBS and detect GFP positive cells with flow cytometry.

The efficiency of homologous recombination repair is indicated by green fluorescent protein GFP. The results are shown in FIG. 4 as E: through cell sorting of a flow cytometer, over-expression of DDUP in HeLa cells can obviously increase GFP positive cells, which indicates that the homologous recombination repair function is enhanced; while in DDUP ^-/- -#1、DDUP ^-/- In the case of- #2 cells, overexpression of DDUP/WT also increased GFP-positive cells; however, when DDUP/T174A was overexpressed, GFP positive cells were not changed compared to the control group; indicating that DDUP can promote homologous recombination repair when DNA is damaged.

(6) The regulation and control of DDUP imbalance on PCNA (proliferating cell nuclear antigen) monoubiquitination are analyzed by co-immunoprecipitation and immunoblotting experiment:

firstly, HeLa/DDUP are added ^-/- -#1、HeLa/DDUP ^-/- - #2 cells were treated at 3X 10 cells, respectively ⁵ The density of/mL was evenly spread in six well plates.

Secondly, after the cells are cultured to about 70% of the density, the empty cells and the DDUP plasmid (DDUP-psin plasmid in (3) of example 1) are transfected into HeLa cells according to the above-mentioned liposome transfection method; DDUP/WT, DDUP/T174A plasmids (DDUP/T174A vector in (5) of example 3) were transfected into DDUP ^-/- -#1、DDUP ^-/- - #2 cells; after 48 hours, CDDP (final concentration of 5. mu.M), UV (60J/M) were used ² ) DNA damage is induced.

③ collect the cells into a 1.5mL EP tube by cell scraping, and wash the cells with 1 XPBS for 2 times; the supernatant was discarded and 200. mu.L of buffer A ((10mM KCl,15mM MgCl) ₂ ,10mM HEPES,pH 7.9,0.34mM sucrose(sucrose), 1mM dithioreitol (dithiothreitol), 10% glycerol (glycerol), 0.1% Triton X-100 and the protease inhibitor cocktail ((# P8340, Sigma-Aldrich)) resuspended cells and incubated on ice for 10 minutes.

Fourthly, centrifuging at 1300g for 5 minutes, discarding the supernatant, and collecting cell sediment.

And fifthly, washing for 2 times by using the buffer solution A.

Sixthly, 200. mu.L of lysate B (0.2mM EGTA,1mM dithiothreitol, and protease inhibitor mix) is added, vortexed for 30 seconds, lysed on ice for 20 minutes, and vortexed repeatedly every five minutes.

Collecting chromatin components at 1700g for 5min at 4 deg.C.

And (iii) rinsing the test specimen with lysate B, resuspending the test specimen with a sample buffer, and performing an immunoblotting experiment.

The results are shown in fig. 4 as F: in a HeLa cell, over-expression of DDUP can obviously increase PCNA of single ubiquitination on chromatin, and meanwhile, a co-immunoprecipitation experiment also shows that PCNA can be combined with ubiquitin, which indicates that DDUP can promote more PCNA to chromatin sites and promote PCNA single ubiquitination when DNA is damaged; in DDUP ^-/- -#1、DDUP ^-/- In the- #2 cell, singly ubiquitinated PCNA on chromatin was significantly reduced upon DNA damage compared to the control; counting the PCNA focus of the cell nucleus, and when no DNA damage occurs (namely 0 hour), the PCNA rarely forms the focus; after 10 minutes of DNA damage, the PCNA focus is rapidly increased and reaches a plateau stage after 30 minutes; then, as time goes on, the PCNA focus slowly decreases, and the PCNA focus returns to the background value after 24 hours; in contrast, in DDUP ^-/- In cells, due to the complete knockout of DDUP, after DNA damage reaches a plateau stage, PCNA focus is rapidly reduced, and the background value is recovered to 12 hours; the above results indicate that DDUP can maintain PCNA focus on damaged chromosomal sites for a long time to promote DNA repair.

Example 5 expression of DDUP can be used as a diagnostic marker for ovarian platinum drug resistance in vitro

(1) Relationship of CTBP1-DT to progression-free survival, survival after progression or recurrence-free survival of tumor patients:

disease-Free Progression analysis (PFS), Progression Survival analysis (Post Progression Free Survival, PPS), recurrence-Free Survival analysis (RFS) were performed on CTBP1-DT using the Kaplan-Meier plotter (https:// kplot. com/analysis /) on-line tool analysis, with the results shown in FIG. 5A: the high expression of CTBP1-DT was associated with poorer survival in patients, whether ovarian, lung, gastric, or breast cancer; as can be seen, CTBP1-DT is a broad oncogene.

(2) Immunohistochemical analysis:

367 of paraffin-embedded platinum-resistant ovarian cancer tissues and 4 of fresh ovarian cancer tissues were used, as well as clinical data from the patients. All the samples are sampled by gynecology of the center for tumor prevention and treatment of Zhongshan university and are sent to the department of pathology of the Hospital for tissue embedding, slicing and diagnosis. Paraffin tissue sections used in this study were provided by the department of Pathology of the Hospital, and the use of the samples had been approved by the patient's informed consent and by the university of Central America ethical Committee. In 367 clinical samples of ovarian cancer based on platinum drug treatment, the expression value of the DDUP protein level is detected by an immunohistochemical experiment, and then relevant clinical information is analyzed and classified according to the analysis result of the 367 clinical samples. Samples were divided into two groups, DDUP high expression group and DDUP low expression group according to the immunohistochemical results, and the correlation between the two groups of samples and Platinum drug Resistance (Platinum Resistance) and recurrence (Relapse) and the overall survival rate and recurrence survival rate of the two groups of samples were analyzed. The specific experimental method is as follows:

firstly, a dyeing step:

a. dewaxing: placing the tissue slices in a constant-temperature incubator at 65 ℃ for baking for 2 hours, immediately soaking in a fresh xylene solution for 10-15 min, and continuously soaking for 10-15 min after replacing fresh xylene;

b. hydration: sequentially soaking the tissue slices in absolute ethyl alcohol, 95% ethyl alcohol, 75% ethyl alcohol and distilled water, and treating each solution for 3-5 min;

c. enzyme removal: soaking the tissue slices in 3% H ₂ O ₂ Inactivating endogenous peroxidase in the solution, and treating at room temperature for 15 min;

d. antigen retrieval: soaking the tissue slices in sodium citrate buffer solution for microwave repair, thawing for 2min with high fire for 5min, thawing with high fire for 20min, taking out, and cooling in a fume hood;

e. and (3) sealing: rinsing the cooled tissue slices for 2-3 times by 1 multiplied by PBS, spin-drying, drawing the edges of the tissues by using an immunohistochemical pen, dropwise adding 100 mu L of closed working solution on the tissues, and treating for 20min at room temperature;

f. a first antibody: throwing off the sealing working solution, dripping 100 mu L of freshly prepared target antibody (DDUP antibody) solution, slightly shaking to fully cover the tissue, and incubating overnight at 4 ℃;

g. washing: wash 3 times with 1 × PBS to remove unbound antibody, 5min each;

h. secondary antibody: spin-drying the tissue slices, then dropwise adding 100 mu L of biotin labeled goat anti-mouse/rabbit IgG solution, and incubating for 20min at room temperature;

i. washing: washing with 1 × PBS for 3 times, 3-5 min each time;

j. connecting: throwing off the secondary antibody solution, dropwise adding 100 mu L of horseradish enzyme labeled streptavidin working solution, and reacting at room temperature for 20 min;

k. washing: washing off redundant connecting liquid by using 1 XPBS, repeating for 3 times, and each time for 3-5 min;

color development: spin-drying the slices, dripping DAB color developing solution prepared within 30min on the tissues, observing under a microscope, and stopping color developing reaction by using distilled water immediately after positive staining of the tissues occurs;

m. washing: after all the slices are developed, rinsing for 3-5 times by tap water;

n. counterdyeing: immersing the tissue slices in a hematoxylin solution for counterstaining for 2-3 min, and rinsing for 5-8 times by using tap water;

o. differentiation: soaking the tissue slice in a hydrochloric acid alcohol solution for 5-10 s, and immediately placing the tissue slice in tap water to terminate the reaction when the rosy of the tissue appears by naked eyes;

p. blue return: washing the slices with tap water for about 30min, and stopping when nucleus is blue;

and q, dehydration: sequentially soaking the tissue slices in 75% ethanol, 95% ethanol and absolute ethanol for gradient dehydration for 3-5 min respectively;

r, mounting: after the slices are fully dried, a proper amount of neutral resin is dripped, a cover glass is covered, and bubbles are removed by lightly pressing the glass slide.

Scoring:

all immunohistochemical staining results in this study were scored independently by two specialized pathologists under double-blind conditions, and the average of the two scores was used as the final score. And the immunohistochemical scoring adopts a semi-quantitative method, and combines two indexes of staining intensity and staining range to judge an expression result. Wherein the staining intensity is scored to be 0-3 points (0: staining is negative, 1: staining is weak positive, 2: staining is positive, and 3: staining is strong positive); the staining range is 0-4 points (0: no positive cell, 1: 10% positive cell, 2: 10% -35% positive cell, 3: 36-75% positive cell, 4: 75% positive cell) according to the percentage of the positive cells, finally multiplying the staining intensity and the staining range score, and the obtained score (namely SI) is the final score, wherein SI is more than or equal to 8, high expression and SI is less than 8, and the obtained score is the final score.

The results of the experiment are shown in B, C in FIG. 5: the staining degree of DDUP is obviously deepened in the platinum drug-resistant tissues, which indicates that the DDUP is related to the drug resistance of ovarian cancer patients; chi fang test shows that the DDUP expression level is related to platinum drug resistance and relapse of patients; Kaplan-Meier survival analysis shows that the total survival time and disease-free survival time of the platinum drug-treated patient with higher DDUP expression level are lower than those of the platinum drug-treated patient with lower DDUP expression level; COX survival analysis shows that the high expression level of DDUP indicates higher death, platinum drug resistance and recurrence, so that the DDUP can be used as an independent factor for judging drug resistance, prognosis and tumor recurrence of tumor patients.

(3) Immunoblot analysis of chromosome-bound and total DDUP expression in patient-derived ovarian cancer cells (PDOVC):

the experimental procedure was the same as (6) in example 4 except that: the cells are isolated from fresh tissue of clinical patient origin; berzosertib (final concentration 80nM) inhibitor with or without ATR; CDDP (final concentration 5. mu.M) was used or not. The results are shown in FIG. 5 at D: compared with PDOVCs #1 and PDOVCs #2, the DNA damage shows higher-level DDUP on chromatins of PDOVCs #3 and PDOVCs #4, which indicates that the PDOVCs #3 and the PDOVCs #4 are resistant to platinum drugs, while the PDOVCs #1 and the PDOVCs #2 are relatively sensitive to the platinum drugs; when CDDP was used in combination with Berzosertib, it was observed that DDUP on chromatin of platinum-drug-resistant samples #3 and #4 disappeared while DDUP in whole cell lysates remained unchanged, indicating that Berzosertib could inhibit DDUP on chromatin.

(4) Homologous recombination repair assay of patient-derived ovarian cancer cells (PDOVC):

the experimental procedure was the same as (6) in example 4 except that: the cells are isolated from fresh tissue of clinical patient origin; CDDP (final concentration 5. mu.M) was used; berzosertib (final concentration 80nM) as an inhibitor of ATR was used or not. The results are shown in FIG. 5, E: compared with PDOVCs #1 and PDOVCs #2, the homologous recombination repair efficiency of the PDOVCs #3 and the PDOVCs #4 is higher during CDDP treatment, so that the method is more tolerant to platinum drugs; in contrast, the repair efficiency of homologous recombination of PDOVCs #1 and PDOVCs #2 is very low; when CDDP was used in combination with Berzosertib, it could be observed that the platinum drug resistant samples #3 and #4 were inhibited from homologous recombination repair, indicating that Berzosertib could inhibit homologous recombination repair by inhibiting DDUP, which is consistent with the experimental results shown in FIG. 5D.

(5) Immunoblot experiments analyzed chromosome binding and expression of total monoubiquitinated PCNA and DDUP in patient-derived ovarian cancer cells (PDOVC):

the experimental procedure was the same as (6) in example 4 except that: the cells are isolated from fresh tissue of clinical patient origin; CDDP (final concentration of 5. mu.M) or UV (60J/M) was used ² ). The results are shown in fig. 5 as F: PDOVCs #3 and PDOVCs #4 exhibit higher levels of monoubiquinated PCNA on chromatin at DNA damage relative to PDOVCs #1, PDOVCs # 2; when CDDP was used in combination with Berzosertib, disappearance of monoubiquitinated PCNA on chromatin from platinum-drug resistant #3 and #4 samples was observed, indicating that Berzosertib could inhibit chromatin monoubiquitinated PCNA.

Example 6 in vivo DDUP can be used as a therapeutic target for ovarian cancer drug resistance

(1) Establishing a patient-derived ovarian cancer xenograft model:

after retrieving fresh clinical patient tissue (PDOC #3 and PDOC #4), the tissue was cut into 20-0 mm pieces after washing twice with PBS in a clean bench, and the tissue pieces were inoculated one site each (as in a in fig. 6) into the ovaries of NOD-SCID IL-2r γ -/- (NSG) mice (jiangsu ji gaokang biotechnology limited) using puncture needles. Two weeks later, the patient tissue-inoculated mice were divided into four groups (6 mice per group), control group, carboplatin group, Berzosertib group, and carboplatin, Berzosertib combination treatment group, each given the corresponding drug therapy:

control (Vehicle) group: performing intraperitoneal injection of DMSO solution at a dose of 100 μ L/time for 3 times/week;

(iii) Carboplatin group: intraperitoneal injection, 50mg/kg, 100 μ L/time, 3 times/week;

group Borzosertib: intraperitoneal injection, 60mg/kg, 100 μ L/time, 3 times/week;

combined carboplatin and Berzosertib group: carboplatin, 50mg/kg, 100 μ L/time; berzosertib, 60mg/kg, 100. mu.L/time; 3 times per week.

After 6 weeks of drug treatment, statistics is carried out on the survival of four groups of mice, tumor tissues in the bodies of the mice are taken out, and the tumor volumes and weights of the tissues are measured; after the tumor tissues in the abdominal cavities of the mice of the above groups were taken out, the sections were embedded, and immunohistochemical analysis and TUNEL immunofluorescent staining were performed. The results are shown in FIGS. 6B to F: in the PDX model, after 6 cycles of drug treatment, carboplatin and Berzosertib are used in combination, so that the weight and the volume of a tumor can be obviously inhibited, the survival period of a mouse is prolonged, and the difference is obviously different when the drug treatment is carried out for 3 weeks; the immunohistochemical result also indicates that the combined treatment of carboplatin and the ATR inhibitor Berzosertib leads to the significant increase of gamma-H2 AX of tumor cells, the significant decrease of cell proliferation index ki67 and the significant increase of the apoptosis ratio. Shows that the combined use of carboplatin and the ATR inhibitor Berzosertib can effectively inhibit the ovarian cancer tumor with high expression of DDUP.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> center for tumor prevention and treatment of Zhongshan university (Zhongshan university affiliated tumor Hospital, Zhongshan university tumor research)

Institute)

Application of DDUP as tumor drug resistance detection, treatment and prognosis molecular target

<130>

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<170> PatentIn version 3.5

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Claims

1. A DDUP, the amino acid sequence of the DDUP comprising:

2. The biomaterial related to DDUP as in claim 1, comprising any one of a1) -a 4):

a1) a nucleic acid molecule encoding DDUP of claim 1;

a2) an expression cassette comprising the nucleic acid molecule of a 1);

a3) a vector comprising the nucleic acid molecule of a 1);

a4) a cell line comprising the nucleic acid molecule of a 1).

3. The application of the substance for detecting DDUP in preparing tumor drug resistance detection products, tumor prognosis detection products or tumor recurrence prediction detection; the amino acid sequence of the DDUP comprises:

4. Use according to claim 3, characterized in that: the medicine in the tumor drug resistance comprises platinum drugs.

5, application of DDUP inhibitor in preparing products for improving the sensitivity of tumors to platinum drugs; the amino acid sequence of the DDUP comprises:

6. Use according to claim 5, characterized in that:

the DDUP inhibitor comprises at least one of a substance inhibiting DDUP activity, a substance degrading DDUP and a substance reducing DDUP expression level;

preferably, the substance inhibiting DDUP activity comprises a substance inhibiting DDUP phosphorylation; further preferably, the substance inhibiting DDUP activity comprises an ATR inhibitor;

b1) siRNA, dsRNA, miRNA, ribozyme, gRNA, or shRNA targeting DDUP;

b2) a nucleic acid molecule encoding b 1);

b3) an expression cassette, vector or transgenic cell line comprising b 2);

preferably, the sequence of the shRNA targeting DDUP is shown as SEQ ID NO.3 or SEQ ID NO. 4;

preferably, the sequence of the gRNA targeting DDUP is shown as SEQ ID NO.5 or SEQ ID NO. 6;

7. A medicament, comprising: a DDUP inhibitor;

the amino acid sequence of the DDUP comprises:

8. The medicament of claim 7, wherein:

the substance inhibiting DDUP activity comprises a substance inhibiting DDUP phosphorylation; further preferably, the substance inhibiting DDUP activity is an ATR inhibitor;

b1) siRNA, dsRNA, miRNA, ribozyme, gRNA, or shRNA targeting DDUP;

b2) a nucleic acid molecule encoding b 1);

b3) an expression cassette, vector or transgenic cell line comprising b 2);

preferably, the sequence of the siRNA targeting DDUP is shown as SEQ ID NO.21 or SEQ ID NO. 22;

preferably, the medicament further comprises a platinum-based drug.

9. A kit, comprising: detecting the substance of DDUP; the amino acid sequence of the DDUP comprises:

Use of an ATR inhibitor for the preparation of a DDUP inhibitor, the amino acid sequence of said DDUP comprising: