The Effect Of MiR-373-5p And MiR-382-5p On Chemoresistance Of Epithelial Ovarian Cancer

Background and objective:Ovarian cancer(OC)is one of the three major malignant tumors of gynaecological oncology.Due to its deep anatomical location,the onset of the disease is concealed.In the early stage,the patient always does not have a symptom,and is diagnosed at a advanced stage.The mortality rate is among the top of the gynecological malignant tumors.The standard treatment of ovarian cancer is the standardized operation with platinum based chemotherapy.With the continuous progress of biological medicine,chemotherapy regimens also continue to improve,so the treatment effect of ovarian cancer also improves.However,the early effective cure patients often return to the hospital after two to three years because of the tumor recurrence.When given chemotherapy drugs again,the patients’chemotherapy effect are not as effective as before.The main reasons lie in the multi-drug resistance(MDR)generated in the ovarian cancer cells.Multi-drug resistant tumor cells crossly resistant to a variety of antitumor drug with different structures,different targets and different mechanisms at the same time,which results in the treatment of tumors in the bottleneck period.microRNAs(miRNA)are small,evolutionarily conserved,non-coding RNAs of approximately 18-25 nucleotides(nt)in length,which are generally involved in post-transcriptional gene regulation.When the complementary matching degree between miRNA and the target mRNA is high,the mRNA gets degradation.When the complementary matching degree is low,the translation of the target mRNA is restrained with the down-regulated protein expression,but no change in mRNA level.Through that,miRNA participated in a lot of biological processes,such as proliferation,differentiation,motility,apoptosis and so on.Numerous studies have shown that aberrant miRNA expressions were found in resistant tumor samples and sensitive samples.The aberrant expression of miRNA has a potential to be a biomarker in early diagnosis and judging the prognosis of ovarian cancer patients.And the miRNA can regulate the sensitivity of tumor cells to chemotherapy drugs by affecting the target genes.In our previous study,we detected the aberrant expression profile of miRNA by using the microarray assay.The results showed that the expression of miRNA was different in ovarian cancer drug resistant tissue and sensitive tissue.Then we verified the expression of miR-373-5p and miR-382-5p by qRT-PCR in30 ovarian cancer drug resistant tissues and 42 sensitive tissues.The results showed that the expression of mi R-373-5p was downregulated and mi R-382-5p was upregulated,which exhibited that the aberrant expression of miR-373-5p and miR-382-5p influenced the sensitivity of ovarian cancer cells to chemotherapy drugs,especially platinum.If we can improve the level of the expression of miR-373-5p or silence the expression of miR-382-5p,the sensitivity of epithelial ovarian cancer cells to chemotherapy drugs can be improved.Therefore,we aim to verify the impact of miR-373-5p and miR-382-5p on the biology function of ovarian cancer cell lines SKOV3-GFP/DDPⅡ(SKDDPⅡ)through the experiments in vitro,and find the target genes of miR-382-5p.Then,MTAP was a candidate target gene of miR-382-5p,its expression in ovarian cancer,and its reaction with platinum based chemotherapy and prognosis was discussed.Last,we evaluated the relationship of miRNA with the prognosis of ovarian cancer by meta analysis.Methods:1 The biological effect of miR-373-5p and miR-382-5p on chemoresistance of epithelial ovarian cancer1.1 We detected the resistant index of drug-resistant cell line SKDDPⅡby the CCK8 method,and the expression of miR-373-5p and miR-382-5p in sensitive cell lines SKOV3-GFP(SKGFP)and resistant cell lines SKDDPⅡwas detected by qRT-PCR.Then we use lentivirus expression vector successfully built the stable SKDDPⅡcell model of up-expressed miR-373-5p and stable down-expressed miR-382-5p.Then we verified the expression of miR-373-5p and mi R-382-5p by q RT-PCR.1.2 After transfection of LV-miR-373-5p and LV-anti-miR-382-5p in SKDDPⅡ,we detected the change of IC50 by CCK8 method,the migration and invasion ability by transwell,the proliferation ability by tablet clone formation test,the cell apoptosis and cell cycle change by flow cytometry.1.3 In vivo,expanding SKDDPⅡcell culture of stable transfection with miR-373-5p and subcutaneously injected into the groin of nude mice.When the tumor size was up to 300-400 mm~3,the nude mice were given cisplatin intraperitoneal injection of 2.5 mg/kg,once every other day,eight times continuously.Observe the changes of the tumor size.2 The verification of the target genes of miR-382-5pWe predict target genes of miR-382-5p by biological software including miRBase,TargetScan,microRNA databases and verified the protein expression of target genes of the cells after transfection by Western Blot.Finally,through the dual luciferase report system,we validate the binding sites between mi RNAs and target genes.3 The effect of mi R-382-5p’s potential target gene MTAP deficiency on the prognosis and platinum resistance in ovarian cancerWe analyzed the association of the the predicted target gene of miR-382-5P with ovarian cancer patients’prognosis by Kaplan–Meier plotter(KM plotter),finally we got MTAP,and validated the MTAP’s prognostic value in OC,Kaplan-Meier survival plot and P value were calculated.Further,we examined the expression of MTAP in OC platinum-resistant cell lines and OC tissues.Then we retrieved and compared the expression of MTAP in 90platinum-resistant and 197 platinum-sensitive ovarian cancers,according to The Cancer Genome Atlas(TCGA)Ovarian Statistics data.Last,the association between MTAP and drug resistance in OC was analyzed by bioinformatic analysis.4 A meta-analysis of miRNA as a prognostic marker in ovarian cancerA comprehensive search of PubMed and EMBASE was performed to identify eligible studies.Overall survival(OS),progression-free survival(PFS)and disease-free survival(DFS)as a prognosis for ovarian cancer were extracted and calculated,if available.Pooled hazard ratios(HRs)and 95%confidence intervals(CIs)were calculated using STATA(version 11.0)Results:1 The biological effect of miR-373-5p and miR-382-5p on chemoresistance of epithelial ovarian cancer1.1 The 50%inhibitory concentration(IC50)of cisplatin in the SKDDPⅡcell lines was about 2.35-fold higher than the parental SKGFP cell lines.Quantitative real-time PCR revealed that miR-373-5p was significantly downregulated in SKDDPⅡcells compared with parental SKGFP cells,and miR-382-5p was significantly upregulated(p<0.05).1.2 The level of miR-373-5p was increased significantly in SKDDPⅡcells after transfected with LV-miR-373-5p overexpression lentivirus compared with the negative control(p<0.05),and miR-382-5p was decreased significantly in SKDDPⅡcells after transfected with LV-anti-mi R-382-5p downexpression lentivirus(p<0.05).1.3 We found that the IC50 of cisplatin was decreased in cells that had been transfected with LV-mi R-373-5p or LV-anti-miR-382-5p compared with the negative control.(p<0.01,p<0.05)1.4 The effect of miR-373-5p and miR-382-5p on the migration and invasion ability of ovarian cancer cells was detected by the transwell assay.The numbers of cell permeating septum were significantly decreased after the infection of recombinant lentivirus LV-miR-373-5p or LV-anti-mi R-382-5p(p<0.01).1.5 The ability of colony formation of the SKDDPⅡcells transfected with LV-miR-373-5p or LV-anti-miR-382-5p was decreased obviously under the effect of 0.4μg/ml cisplatin(p<0.01,p<0.05).1.6 The FCM analysis showed that the apoptosis rate of the SKDDPⅡcells transfected with LV-miR-373-5p or LV-anti-miR-382-5p was significantly higher than the negative control groups under the effect of cisplatin(p<0.05).1.7The FCM analysis also showed that the cell cycle of the SKDDPⅡcells transfected with LV-miR-373-5p or LV-anti-miR-382-5p was arrested in G0/G1 phase significantly under the effect of cisplatin compared with the negative control groups(p<0.05).1.8 The model of downexpressed miR-373-5p ovarian cancer xenografts in nude mouse was established,the results showed that lentivirus mediated miR-373-5p had no significant effect on the growth of xenografts no matter before or after given the treatment of cisplatin compared with the control group,p>0.05.2 The verification of the target genes of miR-382-5p2.1 We predicted target genes of mi R-382-5p by biological software and chose PTEN/HIPK3 as the target genes of miR-382-5p.2.2 We found that the PTEN and HIPK3 protein expression level of anti-miR-382-5p group was higher than the negative control group by Western Blot.2.3 The dual luciferase reporter vectors showed that HIPK3 was a target gene of miR-382-5p,and the possibility of the existing of binding sites between PTEN and miR-382-5p was lower.3 The effect of mi R-382-5p’s potential target gene MTAP deficiency on the prognosis and platinum resistance in ovarian cancer3.1 We analyzed the association of the target gene of miR-382-5p with ovarian cancer patients’prognosis by Kaplan–Meier plotter(KM plotter),finally we got that MTAP and PTEN were closely correlated with the prognosis of ovarian cancer.Since the relation between PTEN and chemoresistance of OC was clear,we discussed the effect of MTAP on prognosis and chemoresistance of OC.Computed by KM plotter,low expression of MTAP was associated with OC patients’poor progression-free survival(p=0.027)and overall survival(p=0.0033).3.2 Compared with the parental OC cell lines,low expression of MTAP was found in platinum-resistant cell lines:A2780/DDP(p=0.021),A2780/CBP(p=0.002)and SKOV3/DDP(p=0.008).3.3MTAP’sdown-expressionwasalsofoundbetween10platinum-resistant and 10 platinum-sensitive tissues(p=0.003).Running data from TCGA,MTAP in platinum-resistant group was down expressed compared with platinum-sensitive group(p=0.024).3.4 Bioinformatic process showed close relationship between MTAP and drug resistance of ovarian cancer.4 A meta-analysis of miRNA as a prognostic marker in ovarian cancerTwenty articles with a total of 2,045 participants were included in this meta-analysis.Pooled hazard ratios(HRs)and 95%confidence intervals(CIs)were calculated,resulting in the pooled HR 2.232,(95%CI:1.607-3.100,p<0.001)for OS,1.02(95%CI:0.45-2.32,p>0.05)for PFS,and 1.94(95%CI:1.30-2.91,p<0.001)for DFS.Conclusions:1.The SKDDPⅡcells after up-expressed miR-373-5p or down-expressed miR-382-5p was arrested in G0/G1 phase to increase the apoptosis of cells under the effect of cisplatin,which enhanced the cellular sensitivity to cisplatin.And up-expressed miR-373-5p or down-expressed miR-382-5p can also decrease the drug-resistant cell migration and invasion ability.2.miR-382-5p affected the chemoresistance of ovarian cancer cells by regulating HIPK3 and downexpression of mi R-382-5p could improve the PTEN expression,but the dual luciferase report system showed the negative result,which implied that miR-382-5p improved the chemosensitivity of ovarian cancer cells by indirectly regulating PTEN.3.These data demonstrate that low expression of MTAP is associated with platinum based chemotherapy resistance and worse prognosis in OC.MTAP could serve as a potential biomarker for prognosis and predictor for platinum based chemotherapy response in OC.4.The meta analysis results indicated that ovarian cancer patients with decreased microRNA expression were associated with shorter OS and DFS.It suggested that microRNAs might be promising markers for predicting the survival rate of ovarian cancer.