The Impact Of MiR-31 And MiR-222 On Chemoresistance Of Epithelial Ovarian Cancer And The Potential Molecular Mechanisms

Objective: Epithelial ovarian cancer(EOC)is the most lethal gynecological malignancy,most patients are diagnosed at advanced-stage.Standard of care for epithelial ovarian cancer is surgery followed by chemotherapy consisting of a platinum based compound in combination with paclitaxel.Most patients with advanced ovarian cancer show initial clinical responses to therapy,but almost all eventually relapse.As a consequence,the 5-year survival rate of advanced-stage epithelial ovarian cancer is only 30%.Several laboratory evidence support the existence of cell populations in ovarian cancer with stem cell features.Cancer stem cells(CSCs)are considered to be the root cause of tumor progression,drug resistance,relapse and metastasis.micro RNAs(miRNAs)are small noncoding RNAs that negatively regulate the expression of target genes by translational repression and/or degradation of mRNA targets.Recent findings suggest that miRNAs might be involved in the self-renewal and differentiation of CSCs.This study intents to explore the role of miR-31 and miR-222 in regulating the chemoresistance of epithelial ovarian cancer and the potential molecular mechanisms.Hopefully,the study will provide clues for the development of new target molecules for prognosis and therapy of ovarian cancer.Methods:1 Ovarian cancer cells were cultured in stem cell culture medium(serum-free Dulbecco's modified Eagle medium/Nutrient Mixture F-12/ methyl cellulose containing basic fibroblast growth factor,epidermal growth factor,B27)for 14 days and it was found that all the ovarian cancer cells aggregated into spheres,we established the SKOV3/self,ES-2/self cell lines by successive spheroid generations;2 The resistance to cisplatin of SKOV3/self cells and COC1/DDP cells were detected by cck-8 methods;3 The differentially expressed miRNAs between cisplatin-sensitive and cisplatin-resistant ovarian cancer cell line pairs(SKOV3 and SKOV3/self,COC1 and COC1/DDP)were examined by real-time PCR;4 We established miR-31,miR-222 overexpression cell lines using lentivirus;5 The IC50 of Cisplatin on SKOV3-miR31,SKOV3-miR222 cell lines were detected by cck-8;6 The proliferation of EOC cells were detected by cck-8 and colony formation assay;7 Self-renewal assay was used to check the spheroids formation ability of EOC cells;8 The wound healing and transwell assay were performed to examine the migration and invasion of EOC cells;9 The putative target genes of miR-31,miR-222 were predicted by miRWalk program;10 We analysed the correlation between miRNA and target gene expression lever of EOC specimens in TCGA and GEO datasets and the association between gene expression level and clinicopathological features and prognosis in EOC patients.The OS and DFS were compared by the log-rank test using a Kaplan-Meier survival curve.Multivariate Cox proportional hazards regression was used to determine the independent factors affecting OS and DFS.11 GSEA is designed to detect coordinated differences in expression of predefined sets of functionally related genes.We performed GSEA using the RNA-seq platform data to identify the pathways that are significantly enriched in genes associated with target genes.Results:1 The IC50(23.63?M)of SKOV3/self cells to cisplatin was 14.8 times that of SKOV3 cells(IC50=1.596?M),the IC50(7.482?M)of COC1/DDP cells to cisplatin was 2.9 times that of COC1 cells(IC50 = 2.566?M).SKOV3/self cells and COC1/DDP cells were more resistant to cisplatin.2 miR-31 and miR-222 were up-regulated by 2.7-fold and 3-fold in SKOV3/self cells,respectively.miR-31 and miR-222 were up-regulated by 2.35-fold and 1.84-fold,respectively,in ES-2/self cells.3 The IC50 to cisplatin,growth rate,migration and invasion ability of SKOV3-miR31 cells and SKOV3-miR222 cells had not changed.4 The clonogenic rate of SKOV3-miR31 cells(9.3%)and SKOV3-miR222 cells(8.6%)was significantly higher than that of SKOV3-TR cells(5.15%)treating with 0.1?M cisplatin(P<0.05),indirectly reflecting the resistance of SKOV3-miR31 and SKOV3-miR222 cells to cisplatin.5 The second spheroids formation rates of SKOV3-miR31 cells(54%)and SKOV3-miR222 cells(45%)was higher than that of SKOV3-TR cells(16.8%)(P<0.05),indicating that miR-31 and miR-222 overexpression enhances the stemness of ovarian cancer cells.6 miR-222 was negatively correlated with the prognosis of ovarian cancer,and the expression of miR-222 was higher in patients with lymphovascular invasion,suggesting that miR-222 may be associated with metastasis of ovarian cancer.7 The expression level of miR-31 was positively correlated with tumor grade,suggesting that miR-31 may be associated with the differentiation of ovarian cancer.8 PBX2,CCNC,JARID2 and SALL2 were negatively correlated with miR-31,DNMT3 B and ALDH1A1 were negatively correlated with miR-222.9 The target genes of miR-31,miR-222 were correlated with tumor stage,tumor grade,vascular invasion and lymphovascular invasion of ovarian cancer,and they were positively correlated with the prognosis of ovarian cancer.10 The target genes regulate Wnt/?-catenin pathway and Notch pathway,suggesting they are related to the stemness of ovarian cancer stem cell.Conclusions: miR-31 and miR-222 were up-regulated in SKOV3/self and ES-2/self cells.miR-222 was negatively correlated with the prognosis of ovarian cancer and was associated with lymphovascular invasion.The expression level of miR-31 was positively correlated with tumor grade.miR-31,miR-222 may maintain the stemness of ovarian cancer cells to promote drug resistance through the inhibition of the target genes.