MiRNA-9 Regulation Of ARID1A And Epithelial Ovarian Cancer Cell Sensitivity To Cisplatin

Objective:Ovarian cancer is the most lethal one in female Genital system tumors,and the 5-year survival rate of advanced ovarian cancer patients is only about 30%^[1].Ovarian cancer is mostly treated by surgery and paclitaxel and platinum-based chemotherapical drugs.However,ovarian cancer patients often appear to be resistant to cisplatin or cisplatin-based chemotherapical drugs,which is an important cause of the low 5-year survival rate in advanced patients.The ARID1A protein is a subunit of the SWI/SNF chromatin remodeling complex.Recent studies have suggested that the ARID1A gene is also a tumor suppressor gene and is mutated in a variety of tumors^[2-4].It is associated with cisplatin resistance but the role of it differ in different tumors.Therefore,exploring the role of ARID1A gene in cisplatin resistance and what contribute to ARID1A regulation in ovarian cancer is of great clinical significance.We studied whether miRNA-9 regulates ARID1A and miRNA-9/ARID1A axis in cisplatin in epithelial ovarian cancer.Methods:1.We first verify the reality of cisplatin resistance in two pairs of epithelial ovarian cancer cells?A2780FC and C30,A2780sigma and A2780cis?using crystal violet staining assay;2.Using western blot to detected the expression of BRCA1 and ARID1A in the two pairs of epithelial ovarian cancer cells above 3.Using TargetScan software searching computational prediction of the miRNAs that regulate ARID1A;Make a microarray of epithelial ovian cancer cells and detect the miRNAs expression,and use Real-time PCR to confer the expression of miR-9 of epithelial ovarian cancer cells above;4.Establish a stable cell line with high expression of miR-9 in A2780FC and A2780sigma,and detecte the ARID1A protein expression in the stable cell lines by western blot,and the value for change of cispaltin resistance after the tetment of various concentration of cisplatin was measure by crystal violet staining assay;5.Transient transfection experiment in vitro:using lipofectamin 3000 reagent transiently transfected antisense-miR-9 in C30 and A2780cis cell lines to reduce the miR-9 expression and use Real-time PCR to verify the level of miR-9 reduction;Using western blot detect the expression of ARID1A protein;measure the value for change of cispaltin resistance after the treatment of various concentration of cisplatin by crystal violet staining assay in antisense-miR-9 treated C30and A2780cis cell lines;6.detect the expression of Cleaved-capase3 protein after transient transfection of mir-9-antisense in C30 and A2780cis cell lines.Results:1.miR-9 was highly expressed in cisplatin resistant cell lines?C30 and A2780cis?,***P<0.01 in cisplatin resistant tissue microarray.There was no significant difference in the expression of BRCA1 protein in 2.two cell lines.The expression of ARID1A protein in cisplatin sensitive cell lines?A2780FC and A2780sigma?was significantly higher than that of cisplatin tolerant cells?C30 and A2780cis?;3.in the cell line?A2780FC and A2780sigma?sensitive to cisplatin?A2780FC and A2780sigma?,the expression of miR-9,the decrease of the expression of ARID1A protein was not obvious,and there was no obvious change in the resistance of the cells.4.after transient transfection of mir-9-antisense,the expression of ARID1A protein in C30 and A2780cis was downregulated.Compared with the control group,there was no obvious change in IC50,but the absolute value of cell number decreased and the expression of Cleaved-capase3protein increased.conclusions:1.miR-9 was highly expressed in the two cisplatin-resistant cell lines C30 and A2780cis?***P<0.01?,and the expression level of ARID1A protein of these two was lower than that of A2780FC and A2780sigma cisplatin-sensitive cell line respectively.After transient transfection of mir-9-antisense,ARID1A protein expression of C30 and A2780cis cell line were upregulated,suggesting that miR-9 may be involved in the cisplatin resistance of epithelial ovarian cancer by regulating the expression of the ARID1A.2.after transient transfection of mir-9-antisense,there was no obvious change in IC50 in C30 and A2780cis cell lines compared with its control group,but the absolute value of the number of cells decreased and the expression of Cleaved-capase3 protein increased,suggesting that miR-9 in the drug resistant cell line may lead to the cisplatin resistance by inhibiting apoptosis.