CN110478484B

CN110478484B - Application of substance for inhibiting JDP2 expression in preparation of antitumor drugs

Info

Publication number: CN110478484B
Application number: CN201910730699.XA
Authority: CN
Inventors: 李隽�; 宋立兵; 曹丽雪; 吴阁艳; 朱金容; 施东妮; 吴新贵
Original assignee: Guangzhou Jieerke Biotechnology Co ltd; Sun Yat Sen University
Current assignee: Guangzhou Jieerke Biotechnology Co ltd; Sun Yat Sen University
Priority date: 2019-08-08
Filing date: 2019-08-08
Publication date: 2021-10-26
Anticipated expiration: 2039-08-08
Also published as: CN110478484A

Abstract

The invention discloses application of a reagent for inhibiting JDP2 expression in preparing a medicine for improving sensitivity of cells to anti-tumor medicines, an anti-tumor medicine and application of a reagent for detecting JDP2 expression quantity in preparing a kit. Provides a new idea for preventing and treating ovarian cancer.

Description

Application of substance for inhibiting JDP2 expression in preparation of antitumor drugs

Technical Field

The invention relates to the field of biology, in particular to application of a reagent for inhibiting JDP2 expression in preparation of a medicament, an anti-tumor medicament and application of a reagent for detecting JDP2 expression quantity in preparation of a kit.

Background

Ovarian cancer is a gynecological malignant tumor with the highest mortality rate worldwide, and seriously threatens the life health of women. Recent data on cancers in 2018 shows that the number of deaths of ovarian cancer is 184,799, and the mortality rate of the ovarian cancer is in a rapid rising trend compared with that of the data in the past year. Because of the lack of obvious early symptoms and the lack of effective diagnostic screening techniques for ovarian cancer, 70% of ovarian cancer patients are already advanced at the time of initial diagnosis (FIGO stages III and IV). Currently, the primary treatment regimen for advanced ovarian cancer is tumor cytoreductive surgery combined with a platinum-based chemotherapy regimen. In recent years, despite the increasing clinical surgical skill and the satisfaction degree of the tumor cytoreductive surgery, the survival prognosis of the advanced ovarian cancer patients is still not improved fundamentally, and the 5-year survival rate is only 30%. According to recent data, ovarian cancer recurrence is the leading cause of death in ovarian cancer patients, with approximately 75% of ovarian cancer patients dying from tumor recurrence, 60% of them due to tumor resistance. Therefore, the elucidation of the molecular mechanism of ovarian cancer drug resistance provides a new scientific basis for tumor recurrence; has obvious clinical significance for overcoming the drug resistance of the tumor and reducing the death of the ovarian cancer patient.

The anti-cancer mechanism of most clinical chemotherapeutic drugs is by inducing DNA damage that leads to tumor cell death. Research shows that DNA damage drugs such as adriamycin, cisplatin, topotecan and the like induce ROS (reactive Oxygen species) to rise sharply, so that irreversible oxidative damage and cell senescence are caused, and even cell death is caused. However, more and more studies have shown that tumor cells can resist the DNA damage effects induced by chemotherapeutic drugs. Cancer cells that survive chemotherapy drug induction develop oxidative stress tolerance by maintaining highly activated levels of antioxidant reduction, which not only activates the ROS scavenging system to cope with oxidative stress but also inhibits apoptosis. Wherein, Glutathione (GSH) is used as an important non-enzyme antioxidant and plays an important role in resisting DNA damage induced by chemotherapeutic drugs in tumor cells. GSH can be used as antioxidant to directly scavenge ROS, or as electron donor of other redox system, and can form a product related to scavenging peroxide in glutathione system with glutathione reductase (GP) and glutathione S-transferase (GST). The report shows that GSH is in high level in various tumors, and promotes malignant progression of the tumors by inhibiting apoptosis, promoting cell metastasis and resisting radiotherapy and chemotherapy, and the GSH reduction system plays an important role in malignant transformation of the tumors. At present, it is not clear by which mechanism tumor cells regulate GSH to be maintained at a highly activated level. Therefore, the specific molecular mechanism of cancer cells for regulating and controlling GSH synthesis is disclosed, the DNA damage effect of GSH on the GSH resistance chemotherapy drugs induced by the GSH resistance chemotherapy drugs is deeply discussed, and the method has important clinical guiding significance for exploring tumor treatment for countering GSH synthesis targets.

Disclosure of Invention

The invention aims to provide application of an agent for inhibiting expression of JDP2 in preparation of a medicament.

The invention also aims to provide an anti-tumor medicament.

The invention further aims to provide application of the reagent for detecting the expression quantity of JDP2 in preparation of a kit.

The technical scheme adopted by the invention is as follows:

use of an agent for inhibiting expression of JDP2 in the manufacture of a medicament for at least one of:

increasing the sensitivity of cells to anti-tumor drugs;

inhibiting the expression of a GSH synthesis-associated gene in a cell;

inhibiting intracellular GSH levels;

promoting intracellular ROS levels;

inhibits PRMT 5-mediated methylation modification.

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

Still further, the chemotherapeutic agent includes, but is not limited to, at least one of topotecan, cisplatin.

Further, the synthesis related gene comprises at least one of SLC7A11, GCLM and GSS.

Further, the agent inhibits JDP2 expression by silencing JDP 2.

Further, the cell is an ovarian cancer cell.

Still further, the ovarian cancer cells are epithelial ovarian cancer cells.

Further, the medicament is used for treating ovarian cancer.

Still further, the ovarian cancer is epithelial ovarian cancer.

An antitumor agent characterized by: contains an agent for inhibiting the expression of JDP2 and a chemotherapeutic drug.

Further, the chemotherapeutic drug includes, but is not limited to, at least one of topotecan, cisplatin.

Use of a reagent for detecting the expression level of JDP2 in the preparation of a kit for at least one of:

detecting tumor drug resistance;

prognosis of the tumor;

tumor recurrence is predicted.

Further, the drug resistance refers to the drug resistance of the tumor cells to the chemotherapeutic drugs.

Further, the tumor is ovarian cancer.

Still further, the ovarian cancer is epithelial ovarian cancer.

The invention has the beneficial effects that:

the JDP2 is found to be obviously highly expressed in ovarian cancer and is inversely related to the overall survival prognosis and recurrence prognosis of tumor patients by mainly analyzing public databases and clinical sample data. JDP2 was shown to promote resistance to ovarian cancer DNA damaging agents such as cisplatin and topotecan by in vivo and in vitro experiments. Further, it was found by JDP2ch pseq data analysis that DNA damage stress induced JDP2 binds to the promoters of the key factors SLC7a11, GCLM, GSS associated with GSH synthesis. JDP2 activates transcription level of key factors related to GSH synthesis, thereby increasing intracellular GSH level, maintaining ROS homeostasis and resisting cell damage caused by chemotherapeutic drugs. Further detection shows that JDP2 recruits PRMT5, thereby recruiting WDR5/MLL methyltransferase complex to promote methylation modification of GSH synthesis-related key factor promoter H3K4me 3. The small molecule antagonist OICR-9429 interacting with WDR5-MLL antagonizes methylation modification, and combined with chemotherapy drug treatment, the regulation of the JDP2/PRMT5 compound on GSH anabolism is inhibited, so that the ROS level in DNA damage is increased to a lethal dose, and cancer cells are effectively treated. In summary, the following steps: JDP 2can be combined with PRMT5 to promote methylation modification of GSH synthesis-related key factor promoter H3K4me3, thereby maintaining high-level GSH and further promoting tumor resistance and tumor recurrence of ovarian cancer. For ovarian cancer cells with high JDP2 expression, the combined use of the WDR5/MLL inhibitors OICR-9429 and cisplatin/topotecan can effectively inhibit the GSH level in tumor cells, so as to maintain high ROS level and promote tumor cell death. The invention provides a new diagnosis and treatment method for ovarian cancer drug resistance.

Drawings

FIG. 1 is public database NCBI/GEO/GSE 38666; GSE 14407; the expression of JDP2mRNA in ovarian cancer tissues and normal ovaries in GSE66957 was further analyzed by Kaplan-Meier for the prognosis of overall survival and recurrence in the high and low expression JDP groups in ovarian cancer patients.

FIG. 2 shows the levels of protein and mRNA expression of JDP2 in 2 ovarian epithelial cells and 8 human ovarian cancer cells detected by Western blotting and Real-time PCR, and further, the expression of JDP2 detected in 146 ovarian cancer tissues and the relationship between the expression of JDP2 and the 5-year overall survival and 5-year recurrence-free survival of ovarian cancer patients were analyzed.

FIG. 3 shows the expression of JDP2 as a function of apoptosis.

FIG. 4 is a graph showing that JDP2 is involved in regulating the expression levels of SLC7A11, GCLM and GSS, thereby promoting GSH and counteracting ROS production.

Fig. 5 shows that JDP2 binds to PRMT5 under DNA damage stress, thereby promoting GSH generation.

FIG. 6 shows the methylation modification of JDP2/PRMT5 promoter to synthesize a key element promoter under DNA damage pressure.

FIG. 7 shows tumor growth and survival in mice inoculated intraperitoneally with different expression groups of JDP2 following topotecan chemotherapy.

FIG. 8 shows that PDX model is established for constructing primary ovarian cancer tissues OV-1 and OV-2, and the treatment effect of cisplatin/topotecan combined OICR-9429 is detected.

Detailed Description

The present invention will be described in further detail with reference to examples. It will also be understood that the following examples are included merely for purposes of further illustrating the invention and are not to be construed as limiting the scope of the invention, as the invention extends to insubstantial modifications and adaptations of the invention following in the light of the principles set forth herein. The specific process parameters and the like of the following examples are also only one example of suitable ranges, and the skilled person can make a selection within the suitable ranges through the description herein, and are not limited to the specific data of the following examples.

The invention will be further elucidated with reference to the following specific embodiments. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions, such as those described in the molecular cloning instructions (third edition), or according to the manufacturer's recommendations.

The inventors analyzed common data (GSE 38666; GSE 14407; GSE66957) of multiple mRNA expression chips for ovarian cancer tissue NCBI GEO data (http:// www.ncbi.nlm.nih.gov/gds /) by bioinformatics method. The mRNA chips of GSE38666, GSE14407 and GSE66957 all contained mRNA data for 12 normal ovarian epithelial cells and 18, 12 and 57 ovarian cancer tissues, respectively. Among them, JDP2mRNA levels were significantly elevated in ovarian cancer tissues (fig. 1A), suggesting that JDP2 may be involved in malignant progression of ovarian cancer.

In order to verify whether the expression up-regulation of JDP2 is closely related to malignant development of ovarian cancer and survival prognosis of patients, the invention further analyzes the relation of JDP2 to ovarian cancer patient prognosis through Keplan-Meier Plotter survival analysis website (http:// kmplot. com/analysis /). Values are expressed as mean ± SD, with P <0.05 considered statistically significant differences. High expression of JDP2 negatively correlated with the prognosis of overall and relapse survival in patients, suggesting a poor prognosis in patients with high expression of JDP2 (fig. 1B). DNA damaging agents are used clinically to treat ovarian cancer, including the first-line drugs cisplatin and carboplatin, and the second-line drugs topotecan, doxorubicin, and gemcitabine. These drugs induce different types of DNA damage, thereby killing tumor cells. The high expression of JDP2 was found to be significantly correlated with the overall survival prognosis and relapse in patients receiving platinum-or topotecan-drug treatment by KM Plotter analysis (fig. 1B), suggesting that the expression of JDP2 is closely correlated with the resistance of ovarian tumor patients against DNA damaging agents.

Based on the above, the inventors conducted systematic and intensive studies on JDP2 discovered by the inventors from multiple levels of molecules, cells, animals and clinical specimens to clarify the molecular mechanism of JDP2 in the development of ovarian cancer.

Example 1 detection of high expression of JDP2 in clinical tissues is closely associated with poor prognosis in patients with ovarian cancer

This example detects JDP2 protein and mRNA expression levels in 2 ovarian cancer epithelial cells (IOSE80, hosipic) and 8 ovarian cancer cells (OV90, OV56, COV644, OVCAR3, COV362, TOV112D, a2780, and SKOV 3). To further verify whether JDP2 expression correlates with malignancy progression and prognosis of patient survival in ovarian cancer, this example examined JDP2 protein expression in paraffin sections of 146 ovarian cancer patient tissues and analyzed the correlation of JDP2 expression with clinical pathological features of ovarian cancer. The primers used for detecting the JDP2 gene at the RNA level were:

JDP2-F：5’-TGGGCTGTCTCTGTCTGTTG-3’(SEQ ID NO：1)；

JDP2-R：5’-GCTCTGTCATCACTCAGGCA-3’(SEQ ID NO：2)。

as a result: the expression levels of JDP2 protein and mRNA were significantly higher in 8 ovarian cancer cells than in 2 ovarian cancer epithelial cells (FIG. 2A, B) suggesting that JDP2 is highly expressed in ovarian cancer, possibly as a carcinogen. JDP2 was expressed in drug-resistant patient tissues significantly higher than non-drug-resistant patient tissues after chemotherapy. Immunohistochemical (IHC) statistical analysis showed that JDP2 levels were significantly associated with chemotherapy resistance (P < 0.001; r ═ 0.37), relapse (P ═ 0.002; r ═ 0.25) and the fido stage (P ═ 0.009; r ═ 0.21), but negatively associated with shorter overall/relapse-free survival, suggesting poor prognosis and shorter survival in patients with highly expressed ovarian cancer of P2 relative to those with low expression of JDP2 (fig. 2C, D, E).

And (4) conclusion:

1) JDP2 is highly expressed in ovarian cancer cells and tissues;

2) JDP2 is highly expressed in ovarian cancer, and its up-regulation is closely related to patient resistance, fido staging and patient relapse.

Example 2: in vitro experiments prove that JDP2 participates in the regulation of ovarian cancer chemotherapy tolerance

In order to study the biological function of JDP2 in ovarian cancer, the present example established stable high-expression and silent JDP2 expression ovarian cancer cell lines in OV90 and OVCAR3, respectively, using retroviral vector systems. Whether JDP2 has the effect of improving the resistance of ovarian cancer cells to chemotherapeutic drugs is detected in OV90 and OVCAR3 ovarian cancer cells. The effect of JDP2 expression on apoptosis and survival of ovarian cancer cells was examined by treatment with the addition of the DNA damaging agent cisplatin to the cells.

As a result: western Blotting detection results show that the research successfully establishes an exogenous high-expression JDP2 and ovarian cancer stable cell line for silencing endogenous JDP2 expression (FIG. 3A). High expression of JDP2 increased the anti-apoptotic ability of ovarian cancer cells to chemotherapeutic drugs (fig. 3B). Meanwhile, a clone formation experiment shows that the high-expression JDP 2can improve the anti-apoptosis and proliferation promoting effects of ovarian tumor cells (figure 3C); the above results indicate that high expression of JDP2 promotes cytotoxicity of ovarian cancer cells against chemotherapeutic drugs.

And (4) conclusion: JDP2 has the effect of improving the resistance of ovarian cancer cells to chemotherapeutic drugs.

Example 3JDP2 high expression regulation glutathione reduction System

This example uses DNA damaging agents (CPT) to induce cellular DNA damage and enriches JDP 2-binding DNA fragments in treated cells and deep sequencing (ChIP sequencing, ChIP-seq) the purified DNA. The ChIP process uses Millipore chromatin coprecipitation kit, purified DNA samples were sent to jinweizhi corporation for library sequencing and deep sequencing using HiSeq 2000 sequencing instruments. The sequenced data used the bowtie2 (version 2.2.9) algorithm to map reads to human genomic sequence (hg 19). ChIP peak calling was fitted using the MACS (version 1.401) algorithm. When ChIP peak falls 4kb (. + -. 2kb) around the Transcription Start Site (TSS) of the gene, it is defined as TSS. When ChIP peak falls 6kb (+ -3 kb) around the gene TSS, it is defined as promoter. Gene ontology (GO clustering) and KEGG pathway analysis were performed using a Network2 Canvas. ChIP-qPCR, Q-PCR, WB, GSH and ROS detection experiments further detect the regulation and control conditions of JDP2 on three genes, namely SLC7A11, GCLM and GSS. Wherein, the primers for detecting the expression levels of the SLC7A11, the GCLM and the GSS by Q-PCR are respectively as follows:

SLC7A11-F:5’-TCTCCAAAGGAGGTTACCTGC-3’(SEQ ID NO：3)，

SLC7A11-R:5’-AGACTCCCCTCAGTAAAGTGAC-3’(SEQ ID NO：4)；

GCLM-F:5’-ATGGTTTAAACAAGGCGCTCCTGGCG-3’(SEQ ID NO：5)，

GCLM-R:5’-GTACCTGCAGGGATTACAGGCATGAGG-3’(SEQ ID NO：6)；

GSS-F:5’-CCTAGCCGGTTTGTGCTAAAG-3’(SEQ ID NO：7)，

GSS-R:5’-TTTCAGGGCCTGTACCATTTC-3’(SEQ ID NO：8)。

as a result: a clear enrichment of JDP2 in the promoter region of the genome (24%) was found by ChIP-seq data analysis of JDP2 (FIG. 4A). In addition, Gene Ontology (GO) clustering of the ChIP-seq data of JDP2 revealed that JDP2 is clearly associated with "Response to drug", "glutaminone metabolic process", "glutaminone biochemical process" and "glutaminone metabolic" signaling pathways, suggesting that JDP2 is involved in regulating GSH metabolism and is involved in drug Response (see fig. 4B). Consistent with this hypothesis, JDP2 was associated with "glutaminone metabolism" and "Platinum drug resistance" by KEGG signal pathway analysis of ChIP-seq data in this study, further suggesting that JDP2 is involved in GSH metabolism and promotes drug tolerance (fig. 4B). By analyzing the enrichment degree, JDP2 is obviously enriched in the promoters of SLC7A11, GCLM and GSS of GSH synthesis related genes (FIG. 4C), suggesting that JDP2 may regulate the transcription levels of the 3 genes, thereby affecting the GSH level. The enrichment degree of JDP2 in the GSH synthesis-associated gene promoter was further verified by ChIP qPCR. Under the condition of DNA damage, ChIP qpCR shows that JDP2 is obviously enriched in a promoter of a synthesis-related gene, and expression of JDP2 is silenced, so that the enrichment degree of JDP2 in the promoter of a GSH synthesis-related gene is obviously inhibited (FIG. 4D). Consistent with this result, JDP2 significantly promoted mRNA and protein expression of SLC7a11, GCLM, GSS under DNA damage, while silencing JDP2 significantly inhibited mRNA and protein expression of SLC7a11, GCLM, GSS (fig. 4E, F). More importantly, high expression of JDP2 significantly increased intracellular GSH levels but inhibited intracellular ROS levels (fig. 4G). Silencing expression of JDP2, however, significantly inhibited intracellular GSH levels and instead promoted intracellular ROS levels. The above experimental results show that, under DNA damage, JDP2 participates in regulating transcription of GSH-related genes, and promotes GSH generation to resist oxidative stress.

And (4) conclusion: JDP2 is effective in resisting oxidative stress and cytotoxicity caused by chemotherapeutic drugs by regulating GSH reduction system.

Example 4: JDP2/PRMT5 mediates epigenetic modification to promote transcriptional activity of JDP2

(1) PRMT5/JDP2 transcriptional Activity

To further explore the transcriptional regulatory mechanism mediated by JDP2 in DNA damage, this example analyzed JDP2 binding to PRMT5 by co-immunoprecipitation. First, the experiment was performed by treating cells with camptothecin (an inhibitor of DNA topoisomerase I), and further, performing a co-immunoprecipitation experiment by an antibody against RPMT 5. Meanwhile, a plasmid of a truncated JDP2 was constructed, and the domain binding between RPMT5 and truncated JDP2 was examined. The expression of PRMT5 is knocked down, and the influence of PRMT5 on GSH, ROS, SLC7A11, GCLM and GSS is detected.

As a result: in the case of DNA damage, binding of PRMT5 to JDP2 was significantly promoted (fig. 5A). The co-immunoprecipitation experiment of PRMT5 and truncated JDP2 demonstrated that PRMT5 binds to the leucine zipper domain of JDP2 (FIG. 5B). It has been reported in the literature that JDP2 promotes deacetylation of histones by recruiting histone deacetylation protein HDAC3, resulting in transcriptional repression. Then, in the case of DNA damage, is PRMT5 competitively bind to JDP2 with HDAC3 and participate in the transcriptional regulation of JDP 2? Co-immunoprecipitation experiments demonstrated that binding affinity of JDP2 to HDAC3 was significantly inhibited at DNA damage (fig. 5C). The expression of PRMT5 is knocked down, so that the transcription level and the protein level of genes related to the regulation of GSH metabolism by JDP2 are obviously inhibited, the generation of GSH induced by JDP2 is inhibited, and the level of ROS induced by DNA damage is increased (FIG. 5D, E, F). Under DNA damage pressure, PRMT5 competes with HDAC3 for binding to JDP2 and promotes GSH production against oxidative stress.

And (4) conclusion:

1) PRMT5 promotes JDP2 transcriptional activation by antagonizing JDP2 binding to HDAC 3.

2) PRMT5/JDP2 is involved in GSH anabolic processes, counteracting ROS production.

(2) PRMT 5-mediated methylation modification promotes transcription of synthetic GSH-associated genes

The PRMT5 has been reported in the literature to promote transcriptional regulation by methylation modification of H3R2me1 and H3R2me2s of histones, and recruitment of WDR5/MLL complex to promote methylation of H3K4me3 of histones. Modification of H3K4me3 of histone also further recruits RNA polymerase II to participate in transcriptional regulation of genes. In the case of DNA damage, the degree of enrichment of H3R2me1, H3R2me2s, WDR5/MLL, H3K4me3, TFIID, POll on the SLC7a11 promoter was examined, and the regulatory effect of knocking down WDR5 on SLC7a11, GCLM, GSS, and GSH and ROS was examined.

As a result: DNA damage significantly up-regulated the enrichment of H3R2me1 and H3R2me2s on the SLC7a11 promoter, and significantly inhibited the enrichment of H3R2me1 and H3R2me2s on the SLC7a11 promoter when β -catenin, JDP2, PRMT5 was knocked-down (fig. 6A), revealing that PRMT5 facilitated the process of activation of GSH by JDP2 through methylation modification of histones. Consistent with this result, DNA damage-induced JDP2/PRMT5 significantly promoted the enrichment of WDR5, MLL1-3, TFIID and Pol II on the SLC7A11 promoter (FIG. 6B, C). Furthermore, knocking down expression of WDR5 significantly suppressed the transcription level of GSH-related genes, and thus the GSH level, but promoted the ROS level (fig. 6D, E). The small molecule inhibitor OICR-9429 of WDR5 combined with chemotherapeutic drugs significantly promoted the sensitivity of tumor cells to chemotherapeutic drugs and promoted tumor cell death (FIG. 6F). The above experimental results prove that PRMT5 promotes the process of JDP2 activating GSH by regulating histone methylation modification, thereby resisting the killing effect of chemotherapeutic drugs.

And (4) conclusion:

1) JDP2/PRMT5 formed a complex that recruited WDR5, MLL promoted methylation of H3K4, thereby promoting methylation modification and transcriptional upregulation on GSH-associated gene promoters.

2) The WDR5 small-molecule inhibitor OICR-9429 obviously antagonizes epigenetic modification caused by JDP2/PRMT5 complex, and is combined with chemotherapeutic drugs to obviously up-regulate the level of ROS and promote tumor cell apoptosis.

Example 5 combination of Small molecule inhibitors with chemotherapeutic drugs is helpful in treating ovarian cancer chemotherapy tolerance

(1) In vivo experiments prove that JDP2 promotes drug resistance of ovarian cancer to topotecan

Topotecan used clinically is a clinical drug after optimization of CPT and is widely used for the treatment of ovarian cancer, lung cancer and other cancers. This example uses an intraperitoneal tumor implantation as a model, and 5X 10⁶Tumor cells with luciferase expression were injected into the abdominal cavity of BALB/C nude mice, 6 cells per experimental group. Using in vivo fluorescenceThe pixilated enzyme imaging system recorded the fluorescence intensity of the tumor. When the tumor fluorescence value in the mice reaches 2X 10⁷p/sec/cm²Per, treatment was started 3 times per week with topotecan (10mg/kg body weight) for 6 weeks. When the mice were raised to the end of the experiment, all mice were sacrificed by cervical dislocation. Tumor tissues were removed for subsequent experiments and were fixed with formaldehyde and paraffin-embedded sections were used for immunohistochemical staining of the protein of interest.

As a result: the effect of JDP2 dysregulation on DNA damage stress was examined by intraperitoneal ovarian cancer mouse model. As shown in figures 7-a-D, cells up-regulating JDP2 expression in topotecan-treated mice developed tumor maintenance with higher growth rates, as shown by fewer TUNEL + cells, and exhibited higher GSH concentrations, but lower ROS levels, leading to poorer prognosis of survival in mice. In contrast, incorporation of Dioleoylphosphatidylcholine (DOPC) nanoliposomes by short interfering RNA (siRNA) silencing JDP2 significantly enhanced the antitumor effect of topotecan, resulting in lower tumor growth rates and reduced intratumoral GSH levels, but higher ROS levels and apoptosis (FIG. 7A, B, C, D). Thus, the above results further support the notion that JDP2 is involved in regulating GSH metabolism and inhibiting ROS-induced apoptosis to develop resistance to DNA damage therapy.

And (4) conclusion: in vivo experiments, high expression of JDP2 enhanced chemotherapy resistance of ovarian cancer cells to topotecan.

(2) In vivo experiments prove that JDP2 promotes drug resistance of ovarian cancer to topotecan

The present invention employs patient-derived xenotransplants (PDX). The PDX model is obtained by shearing fresh ovarian cancer tumor tissue which is removed just after operation into tumor blocks with the diameter of 1-3 mm, and inoculating the tumor blocks into female NOD-SCID IL-2r gamma^-/-(NSG) mice subcutaneously. Tumor length and width were measured weekly and recorded, and tumor volume (L W) was calculated²/2) and plotting the growth curve of the tumor according to the tumor volume and the recording time when the tumor volume is 0.2cm³At the time, the mice were divided into 6 groups, and each received the solvent, cisplatin (5mg/kg body weight), topotecan (b.p.)10mg/kg body weight), or OICR-9429(3mg/kg body weight) and topotecan (10mg/kg body weight), or OICR-9429(3mg/kg) and cisplatin (5mg/kg body weight), for 3 times per week for a total of 6 weeks. When the mice were raised to the end of the experiment, all mice were sacrificed by cervical dislocation. Tumor tissues were removed for subsequent experiments and were fixed with formaldehyde and paraffin-embedded sections were used for immunohistochemical staining of the protein of interest.

As a result: the present invention further examined the therapeutic effect of the combination of OICR-9429 and DNA damaging chemotherapeutic agents on cancer using two PDX models established in clinical ovarian cancer tissues (FIG. 8-A). As shown in FIGS. 8-A-C, OICR-9429 alone did not significantly inhibit tumor growth, but OICR-9429 treatment in combination with topotecan significantly enhanced the sensitivity of ovarian cancer to topotecan, resulting in high levels of ROS and inhibition of tumor growth. Furthermore, the use of OICR-9429 in combination with topotecan significantly enhanced the sensitivity of ovarian cancer to topotecan, resulting in high levels of ROS and inhibition of tumor growth. Consistent with this result, the use of OICR-9429 in combination with cisplatin significantly enhanced the sensitivity of ovarian cancer to cisplatin, resulting in high levels of ROS and inhibition of tumor growth. Thus, these results indicate that the combined use of classical DNA-damaging chemotherapeutic agents and oic r-9429 provides a new therapeutic strategy for tumor-resistant therapy by promoting high levels of ROS and thereby killing tumor cells.

And (4) conclusion: OICR-9429 inhibits JDP2/PRMT 5-mediated epigenetic modification in the PDX model, and has stronger chemotherapy resistance when used in combination with topotecan/cisplatin.

In conclusion, JDP 2can be used as a tumor drug resistance detection target, a tumor prognosis detection target and a tumor recurrence prediction detection target. Therefore, corresponding tumor drug resistance detection kits, tumor prognosis detection kits and tumor recurrence prediction detection kits can be prepared, and the kits contain a reagent for detecting the RNA transcription level of JDP2, a reagent for quantitatively detecting the protein expression level of JDP2 and the like. The RNA and protein expression level of JDP2 in the tumor tissue of a patient was examined to determine whether the patient was chemotherapy-resistant to topotecan/cisplatin treatment. By detecting the expression of JDP2 in the tumor tissue of the patients, whether the patients have high recurrence tendency is judged. The expression of JDP2 in tumor tissues of patients is detected, so that the combined treatment scheme of WDR5 inhibitor and topotecan/cisplatin is prepared for the patients with high JDP2 expression, and the treatment effect is realized.

It will be readily understood by those skilled in the art that the foregoing is only a preferred embodiment of this invention and is not intended to limit the invention, and that any modification, equivalent replacement or improvement made within the spirit and principle of the invention will fall within the protection scope of the claims.

SEQUENCE LISTING

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GUANGZHOU JIEERKE BIOTECHNOLOGY Co.,Ltd.

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Claims

1. Use of an agent for inhibiting expression of JDP2 in the manufacture of a medicament for at least one of:

increasing the sensitivity of cells to anti-tumor drugs;

inhibiting the expression of a GSH synthesis-associated gene in a cell;

inhibiting intracellular GSH levels;

promoting intracellular ROS levels;

inhibiting PRMT 5-mediated methylation modification;

the agent inhibits expression of JDP2 by silencing JDP2 or a small molecule inhibitor, the agent silencing JDP2 is siRNA, and the small molecule inhibitor is OICR-9429;

the anti-tumor drug is a chemotherapeutic drug, and the chemotherapeutic drug is topotecan or cisplatin;

the cell is an ovarian cancer cell;

the medicament is used for treating ovarian cancer.

2. Use according to claim 1, characterized in that: the synthesis related gene comprises at least one of SLC7A11, GCLM and GSS.

3. An antitumor agent characterized by: contains an agent for inhibiting expression of JDP2 and a chemotherapeutic agent;

the chemotherapeutic drug is topotecan or cisplatin.

4. Use of a reagent for detecting the expression level of JDP2 in the preparation of a kit for at least one of:

detecting tumor drug resistance;

prognosis of the tumor;

predicting tumor recurrence;

the reagent for detecting the expression level of JDP2 is a primer, and the sequence of the primer is shown in SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;

the tumor is ovarian cancer;

the drug resistance refers to the drug resistance of tumor cells to chemotherapeutic drugs;

the chemotherapeutic drug is topotecan or cisplatin.