Investigation Of The Role Of Mirnas In Acquiring Drug Resistance And Their Mechanism In Ovarian Cancer Cells

Background: A better understanding of the mechanisms that underlie this drug resistance should allow treatment to be optimized. Despite the increase in the number of drug-resistance mechanisms that have been described in vitro, so far none of these mechanisms has been shown unequivocally to be important in the clinical setting. Therefore we established an acquired drug resistance model of nude mices through three generation treated with cisplantin sequentially that had been more simulative for the clinical scenario of ovarian cancer. In order to investigate what miRNAs and genes involved in drug resistant. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This study is to investigate the promote effects and mechanism of miR-125b involved in the development of chemoresistance in ovarian cancer.Methods: We using ovarian cancer SKOV3 cell lines that had been adapted for growth in suspension and then injected these cells subcutaneously to develope a mouse xenograft model of ovarian cancer .Consecutively passaging ovarian cancer cells in BALB/C nu mice treated with Cisplantin .Cells from the third passage (SK-G3) were cultured in matrgel to generate mammospheres。And we using CCK-8 assay to detect the cell proliferation and Cisplatin–induced cytotoxicity in SK-G3 cells and their parent SKOV3 cell lines。And using miRNA and cDNA microarray to screening the differentially expressed genes between SK-G3 cells and their parent SKOV3 cell lines。On the other hand,the different expression of miR-125b in Cisplatin-sensitive ovarian cancer cell line(OV2008) and its resistant variant (C13*) were identified by realtime PCR. Functional studies such as cck-8 assay, flow cytometry, were carried out to detect the Cisplatin resistance effect of miR-125b and Bak1 on cells. Real time PCR, western blotting and Luciferase Reporter Assay were used to detect Bak1 is a target of miR-125b. Results: Relative to its parental SKOV3 cells, SK-G3 cells are more proliferative,more resistant to cisplatin ,and have higher sphere formation ability. MicroRNA and whole genome expression microarray results reveal that there are many differentially expressed genes between the tissue of the parental SKOV3 con Xenograft tumors and the SK-G3 Xenograft tumors. On the other hand, by comparing C13* cells with OV2008 cells, we found the levels of miR-125b increased. We also found the upregulation of microRNA-125b causing a marked inhibition of Cisplatin–induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Cisplatin in ovarian cancer cells.Moreover, Bak1 is a direct target of miR-125b, down-regulation of Bak1 suppressed Cisplatin -induced apoptosis and led to an increased resistance to Cisplatin.Conclusions: Our study of the establishment of acquired drug resistance model through consecutively passaging ovarian cancer cells in nude mices treated with cisplatin is successful and this model selected the miRNAs and genes that may be involved in cisplatin resistance. On the other hand,our study indicated that miR-125b had a significant promote effect on chemoresistance of c13* cells and upregulation of miR-125b expression contributes to Cisplatin resistance through suppression of Bak1 expression. These findings have important implications in the development of targeted therapeutics for overcoming Cisplatin resistance in ovarian cancer.