| Ovarian cancer is one of the most common malignant tumors of the female reproductive system,and which is the fourth leading cause of cancer death in women.Because of its insidious onset and lack of effective early detection markers,ovarian cancer is the the highest mortality rates among all gynecological malignancies and most patients are diagnosed with late stage disease[1-3].In 2018,an estimated 295,414 new cases of ovarian cancer will be diagnosed and 184,799 women will die from this disease all around the world[4].It is worth noting that about 75%of patients are in stage ? or ? according to FIGO stage[5]At present,the standard treatment for ovarian cancer is tumor-cell surgical debulking combined with platinum-based combination chemotherapy.However,the vast majority of these women will have their cancer recur within 12 to 24 months after diagnosis and will die of progressively chemotherapy-resistant tumors[6.7].With the application of PARP inhibitors,it has brought landmark progress in the treatment of platinum resistant recurrent ovarian cancer patients,and making a new breakthrough in the treatment of ovarian cancer[8].However,the treatment of drug-resistant ovarian cancer is still the difficulty and research hotspot.It has a great significance to improve the survival rate of patients with ovarian cancer by exploring the molecular mechanism and overcoming of cisplatin resistanceProgrammed cell death 1(PD-1),as the main cell surface receptor of the CD28 superfamily,is one of the major inhibitory molecules to reduce T cell activation[9,10].Its main ligand molecule PD-L1 is expressed on antigen-presenting cells and tumor cells[11,12]?Under physiological conditions,PD1 signaling interferes with T-lymphocyte activation and can result in T-cell anergy or lymphocyte apoptosis.PD-1 on the surface of lymphocytes can be combined with its ligand PD-L1 to inhibit lymphocyte function to prevent autoimmune damage.Under pathological conditions,PD-1/PD-L1 signal pathway is activated,PD-L1 on the surface of the cancer cell interact with receptors on the surface of T cell to inhibit the activity of T cells and avoid the killing by T cells,leading to immune evasion.It have found that the high expression of PD-L1 is related to the poor prognosis of malignant tumors in clinical research[11-15].Patients with high PD-L1 expression show significantly worse overall survival compared with patients with low PD-L1 expression[16].The expression of PD-L1 was up-regulated in non-small cell lung cancer cells after treatment with Platinum,and knocking down PD-L1 can significantly increase the sensitivity of non-small cell lung cancer cells to cisplatin[17].In addition,it has reported that PD-L1 overexpression is associated with poor prognosis in patients with ovarian cancer[18].However,there are no reports on the role and specific mechanisms of PD-L1 in cisplatin resistance in ovarian cancer.MicroRNA(mi-RNA)is a type of 20-25 non-coding small RNA molecules that play an important role in various pathological processes by silencing or degrading its target mRNA molecule to regulate gene expression[19].In recent years,with the in-depth study of tumor drug resistance,the mechanism of miRNA involvement in tumor drug resistance has attracted widespread attention from researchers[20,21].It has been reported that miR-34a-5p was down-regulated in ovarian cancer[22],and up-regulation of miR-34a-5p in medulloblastoma,bladder cancer and ovarian cancer cells could increase the sensitivity to cisplatint[23-25.]In addition,it has been reported that miR-34a-5p could target and regulate the expression of PD-LI in NSCLC and AML,and overexpression of miR-34a-5p effectively negatively regulated the expression of PD-L1[26,27].It has not been reported whether miR-34a-5p was involved in the regulation of cisplatin sensitivity in ovarian cancer by regulating the expression of PD-L1,which needs further confirmation.This study is based on the following three parts.In the first part,the high expression of PD-L1 in chemotherapy resistant ovarian tissues and resistant cells were detected by clinical tissue specimens and ovarian cancer cell experiments,and its participation in the regulation of cisplatin resistance of ovarian cancer was obtained.In the second part,it was found that the expression of miR-34a-5p was decreased in cisplatin resistant ovarian cancer cells,Overexpression of miR-34a-5p could effectively inhibit the expression of PD-L1 and increase the sensitivity of cells to cisplatin.There was a targeted regulatory relationship between them.In the third part,rumor-forming experiments in nude mice were conducted to further verify that silencing miR-34a-5p could significantly promote the growth of tumor in vivo and increase the expression of PD-L1,so as to obtain the negative regulation between PD-L1 and miR-34a-5p.Part I Expression of PD-L1 in ovarian cancer tissues and its role in cisplatin resistant cells of ovarian cancerObjective:To detect the expression difference of PD-L1 in ovarian benign tumor,chemotherapy sensitive and drug-resistant ovarian cancer tissues,as well as in ovarian cancer cell lines SKOV3,A2780 and DDP resistant ovarian cancer cells,and to explore the effect of PD-L1 expression on cell proliferation,cycle,apoptosis and chemotherapy sensitivity.Methods:1.To collect the pathological sections and clinical data of ovarian benign tumor tissue,chemotherapy sensitive ovarian cancer tissue and chemotherapy resistant ovarian cancer tissue as the research object.The expression level of PD-L1 protein was detected by IHC,and PD-L1 mRNA was detected by RT-qPCR in ovarian cancer tissue.The expression level of PD-L1 in chemotherapy sensitive ovarian cancer patients and chemotherapy resistant ovarian cancer patients was analyzed to discuss its clinical significance.2.Ovarian cancer cell lines SKOV3 and A2780 were selected and cultured by continuous gradient increase of cisplatin(DDP)to construct ovarian cancer DDP resistant cell lines(SKOV3/DDP,A2780/DDP).Different concentrations of DDP were used to treat SKOV3,A2780 and DDP-resistant cell lines(SKOV3/DDP,A2780/DDP)to detect the proliferation activity by MTT assay,and calculate the IC50 values.3.The expression of PD-L1 in ovarian cancer cell lines SKOV3,A2780 and DDP resistant cell lines(SKOV3/DDP,A2780/DDP)was detected by RT-qPCR and Western blot,respectively.4.Construction of shRNA-PD-L1 vector,and the biological properties of drug-resistant cells were examined after PD-L1 knockdown by transfection shRNA-PD-L1 vector.Cultured with different concentrations of DDP,MTT assay was used to detect the cell proliferation activity.Flow cytometry was used to detected the cell cycle and apoptosis after knocking down PD-L1 expression.5.Western blot was used to detect the changes of related proteins expression of in cell cycle,apoptosis and drug resistance after PD-L1 expression silenced.Result:1.The immunohistochemical stainings showed that the expression level of PD-L1 in chemotherapy resistant ovarian cancer tissues was significantly higher than that in chemotherapy-sensitive group and ovarian benign tumor group.The results of RT-qPCR showed that,compared with ovarian benign tumor tissues,PD-L1 mRNA expression was up-regulated in both chemotherapy-sensitive ovarian cancer tissues and drug-resistant ovarian cancer tissues,and the expression level of PD-L1 in drug-resistant ovarian cancer tissues was higher than that in chemotherapy-sensitive ovarian cancer tissues.This suggests that PD-L1 overexpression may be involved in ovarian cancer drug resistance.2,This study successfully established cisplatin(DDP)resistant ovarian cancer cell lines(SKOV3/DDP,A2780/DDP).MTT assay showed that the proliferation activity of cisplatin-resistant cell lines was higher than the parent strain,and the IC50 value was also higher.3.The results of RT-qPCR and Western blot showed that the expression of PD-L1 were increased in DDP resistant ovarian cancer cells.4.After transfection shRNA-PD-L1 vector,drug-resistant cells SKOV3/DDP and A2780/DDP were more susceptible to DDP,the IC50 values were reduced.Under the same concentration of DDP,the proliferation activity of DDP-resistant cell lines was significantly reduced,G1 phase cells and the apoptosis rate were increased.5.Western blot analysis showed that knockdown of PD-L1 reduced the expression of multidrug resistance protein 1(MRP1)and cyclin D1 in DDP resistant cells,and the expression of cleaved-caspase-3 and cleaved-PARP were significantly increased.Conclusion:PD-L1 is highly expressed in ovarian cancer resistant tissues,SKOV3/DDP and A2780/DDP cells,and is involved in the regulation of cisplatin resistance of SKOV3/DDP and A2780/DDP cells.Part? MiR-34a-5p regulates drug resistance of ovarian cancer cells by targeting PD-L1Objective:To explore the targeting relationship between miR-34a-5p and PD-L1,and to study the effect and mechanism of miR-34a-5p on SKOV3/DDP and A2780/DDP cells resistance to DDP by regulating the expression of PD-L1.Methods:1.The expression of miR-34a-5p in SKOV3,A2780,SKOV3/DDP and A2780/DDP cells were detected by RT-qPCR.2.The expression plasmids of miR-34a-5p mimic and miR-34a-5p inhibitor were constructed and transfected into SKOV3/DDP and A2780/DDP,SKOV3 and A2780 cells respectively.RT-qPCR was used to detect miR-34a-5p expression to verify the transfection efficiency.RT-qPCR and Western blot were used to detect the level changed of PD-L1 expression.3.PD-L1 mRNA 3'-UTR double luciferase reporter gene plasmid and its point mutation plasmid were constructed and co-transfected with miR-34a-5p mimic to 293T cells respectively to detect the change of luciferase activity and verify miR-34a-5p targeted regulation PD-L1 expression.4.The miR-34a-5p mimic and PD-L1 over-expression vectors were respectively or co-transfected into SKOV3/DDP cells,and cultured by DDP.The proliferation activity of cells in each group was detected by MTT assay,the change of cell cycle and apoptosis rate were measured by flow cytometry.5.The miR-34a-5p mimic and PD-L1 over-expression vectors were respectively or co-transfected into SKOV3/DDP cells,and cultured by DDP.The expression levels of PD-L1,MDR1,cyclin D1 and cleared-caspase-3 and cleared-PARP proteins were detected by Western blot.Result:1.Compared with SKOV3 and A2780 cells,miR-34a-5p was significantly lower in SKOV3/DDP and A2780/DDP cells.2.The results of RT-qPCR and Western blot showed that the expression of PD-L1 was decreased after transfected with miR-34a-5p mimic and the expression of PD-L1 increased after transfected with miR-34a-5p inhibitor,the differences were statistically significant.3.The results of double fluorescent reporter gene showed that PD-L1 was the target gene of miR-34a-5p,and miR-34a-5p targeted negatively regulated the expression of PD-L1.4.The results of MTT assay showed that overexpression of miR-34a-5p mimic decreased the proliferation activity of SKOV3/DDP cells,and the IC50 value was reduced,the apoptosis rate was increased.While overexpression of PD-L1 reversed the inhibition of miR-34a-5p mimic,and the IC50 value of SKOV3/DDP cells was increased significantly,the apoptosis rate was reduced.5.Flow cytometry was used to detect cell cycle.The results showed that overexpression of miR-34a-5p mimic inhibited the cell cycle of SKOV3/DDP in DDP culture,the percentage of G1 phase cells was increased,the percentage of cells in S phase and G2 phase were decreased.Overexpression of PD-L1 reversed the inhibitory effect of miR-34a-5p mimic on cell cycle,decreased the percentage of G1 phase cells,and increased the percentage of S and G2 phase cells.6.Annexin V-FITC/PI staining method was used to detect apoptosis.The results showed that the apoptosis of SKOV3/DDP cell was enhanced after overexpression of miR-34a-5p mimic cultured by DDP.At the same time,overexpression of PD-L1 can reverse the apoptosis of cells and decrease the apoptosis rate.7.The protein expression of MDR1,cyclin D1 were decreased and cleaved-caspase-3 and cleaved-PARP were increased in SKOV3/DDP with overexpression of miR-34a-5p mimic.Meanwhile,overexpression of PD-L1 reversed the role of miR-34a-5p mimic on apoptosis and the protein expression of MDR1,cyclin D1,cleaved-caspase-3 and cleaved-PARP.Conclusion:MiR-34a-5p reverses the cisplatin resistance of SKOV3/DDP by targeting down-regulation of PD-L1.Part? MiR-34a-5p regulates cisplatin resistance of ovarian cancer by targeting PD-L1 in vivoObjective:To investigate the mechanism of miR-34a-5p enhancing the sensitivity of SKOV3/DDP to DDP by targeting down-regulation the expression of PD-L1.Methods:1.Constructed of miR-34a-5p stable overexpression or knockdown vector and transfected into SKOV3/DDP cells.RT-qPCR was used to detecte the expression of miR-34a-5p to demonstrate transfection efficiency,and the protein expression of PD-L1 was detected by Western blot.2.Four week old healthy BALB/C nude mice were divided into 5 groups,10 mice in each group.1 × 106 SKOV3/DDP cell suspensions of each group were injected subcutaneously into the subcutaneous tissue of the right armpit of nude mice to establish the transplanted tumor model,and measured the tumor volume.3.When the tumor volume reaches a certain volume(50 mm3),intraperitoneal injection of 2.0 mg/kg cisplatin for 4 weeks,every 3 days.The tumor size was measured on the 6th day and the tumor growth curve is drawn.After the mice were sacrificed,the tumor size and weight of nude mice were measured.4.The tumor tissue of each group of mice was extracted,and the apoptosis level was detected by TUNEL staining.The expression of miR-34a-5p and PD-L]mRNA was detected by RT-qPCR,and the expression PD-L1 protein and the related proteins of cell cycle,apoptosis and resistance were detected by Western blot.Results:1.Successfully established miR-34a-5p stably over-expressed or knocked down SKOV3/DDP cells,and constructed the model of ovarian cancer transplantation in nude mouse.2.Compared with the other 4 groups,miR-34a-5p significantly enhanced the chemosensitivity of SKOV3/DDP cells to DDP,the transplanted tumor of over-expressed miR-34a-5p nude mouse were smaller in size and lighter in weight.The silencing of mir-34a-5p expression promotes transplanted tumor growth in vivo and enhances the resistance to DDP.3.Overexpression of miR-34a-5p significantly reduced the protein expression of PD-L1,MDR1,CyclinDl and Bcl2,and enhanced the expression of Bax and cytochrome C in tumor tissues.The silencing of miR-34a-5p expression enhanced the protein expression of PD-L1,MDR1,CyclinD1and Bcl2,and the expression of Bax and cytochrome C were reduced.4.TUNEL staining results also showed that overexpression of miR-34a-5p apparently promoted the apoptosis of transplanted tumor tissue cells.However,the effect of silence miR-34a-5p was opposite.Conclusion:MiR-34a-5p enhanced the sensitivity of subcutaneous ovarian cancer xenografts to cisplatin by targeting knocking down PD-L1.The innovation of this study1.The experimental results showed that the up-regulation of PD-L1 expression patients was related to chemotherapy resistance in tumor tissues of resistance ovarian cancer.2.Experimental results of ovarian cancer resistant cells and animal models in vivo showed that miR-34a-5p could reverse the resistance of ovarian cancer.3.MiR-34a-5p targets to regulate PD-L1 negatively,and miR-34a-5p/PD-L1 molecular axis can be used as a potential target for clinical treatment of cisplatin resistant ovarian cancer. |
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