Effect Of MiR-186/Twist1 On EMT Reversal And Cisplatin Resistance In Epithelial Ovarian Cancer Cells

Ovarian cancer has the highest mortality rate among gynecological malignancies,epithelial ovarian cancer(EOC) is the most common histological type, and the major treatment plan for patients is the surgery combined with cisplatin-based chemotherapy,which accompanied by treatment failure for the main reason of the generation of multi-drug resistance(MDR) in chemotherapy. Therefore, study on the mechanism of MDR has become the hotspot and difficulty in the research of prevention and treatment of ovarian cancer.Twist1 plays an important and a dual role in tumor development, on the one hand,it can induce the produce of tumor cells and further maintain epithelial-mesenchymal transition(EMT) phenotype, so as to promote tumor invasion and metastasis; on the other hand, it can promote cell proliferation and inhibit cell apoptosis. In addition, the role of Twist1 in cancer therapy resistance has gradually attracted attention and become the focus of cancer research in the field of tumor. However, its role and regulatory mechanism in EMT and cisplatin resistance in ovarian cancer is unclear.In recent years, regulation of mi RNAs involved in tumor resistance and EMT phenotype is widely recognized. Based on above analysis,the present study was designed to clarify the potential role and molecular mechanism of mi R-186 involved in cisplatin resistance and EMT phenotype in EOC cells by Twist1 regulation, for providing new ideas to reverse multiple malignant phenotype of EOC cells, which is of great significance in the prevention and treatment of ovarian cancer.Part I. Identification of EMT phenotype and the involvement of Twist1 in CDDP-resistant ovarian cancer cellsThe microscope was used to observe the CDDP resistant and sensitive cell morphology. WB was used to detect the protein levels of Twist1 and EMT marker molecules, to determine whether CDDP resistant EOC cells exhibits EMT phenotype.Twist1 interference vector was constructed, and then transfected into CDDP resistant cells to inhibit Twist1 expression. Real-time quantitative PCR(quantitative realtime PCR, q PCR) and WB were used to detect Twist1 m RNA and protein levels respectively. Wound healing assay was used to detect the ability of cell migration. Cell morphology and changes of EMT marker moleculars were observed. CCK-8 assay was used to detect the cell proliferation and calculate IC50 values for CDDP in EOC cells.FCM was used to to detect cell cycle, cell apoptosis.Our data demonstrated that CDDP-resistant cells appeared in mesenchymal-like cell morphology with high expression of mesenchymal marker protein(Vimentin) and low expression of epithelial marker protein(E-cadherin). Twist1 highly expressed in CDDP-resistant cells. Twist1 inhibition can reverse the EMT phenotype accompanied with increasing E-cadherin and decreasing Vimentin, inhibit cell cycle progression,reduce cell proliferation, induct cell apoptosis and increase the sensitivity of EOC cells to CDDP.Part II. Role and regulation mechanism of mi R-186 on EMT phenotype and CDDP resistance in EOC cellsmi R-186 expression levels in CDDP resistant and sensitive cells was detected by q PCR. The correlation between mi R-186 and Twist1 expression was analyzed. Dual luciferase reporter gene system was used to verify whether Twist1 was the target of mi R-186. CDDP resistant cells were transfected mi R-186 mimics(mimics) with or without Twist1 recombinant vector, and CDDP sensitive cells were transfected with mi R-186 inhibitor(mi R-186-INH), then observe the expression change of mi R-186,Twist1 and their downstream target gene, and also the cell morphology, changes of EMT marker molecular, especially the cell migration, proliferation, cell cycle, cell apoptosis and changes in IC50 values for CDDP were detected before and after the treatment. Tumor-bearing nude mice model was established to observe the role of mi R-186 and Twist1 on drug resistance of EOC cells in vivo.We detected that mi R-186 was decreased in CDDP-resistant cells, and negatively correlated with Twist1 expression. Twist1 was a direct target of mi R-186.Overexpression of mi R-186 can reduce Twist1 levels, impair inhibitory effect of Twist1 on p53 that lead to the reversal of EMT phenotype, reduce the cell proliferation, inhibit cell cycle progression, induct the cell apoptosis and increase sensitivity of EOC cells to CDDP, and vice versa. However, effect of mi R-186 on EMT reversal and chemotherapy sensitization can be weakened by Twist1 transfection. The above conclusions were further confirmed by experiments in vivo.Part III Relationship between mi R-186 and EOC drug resistanceExpression of mi R-186 and Twist1 in EOC tissue samples were detected to analysis their correlation, the relationship of mi R-186 or Twist1 levels with chemotherapy sensitivity and Progression-free survival(PFS) of EOC patients were alsoanalyzed respectively.Clinical analysis found: mi R-186 expression negatively correlated with Twist1 in EOC tissues, high mi R-186 level or low Twist1 expression indicates chemosensitivity,and a longer PFS for EOC patients, while low mi R-186 level or high Twist1 expression indicates chemotherapy-resistant, and a shorter PFS for EOC patients.Taken together, this study presents that EOC cells acquired resistance, associated with and maintained EMT phenotype. Twist1 is able to induce EMT and increase CDDP resistance of EOC cells. Twist1 is a direct target of mi R-186. Overexpression of mi R-186 can reduce Twist1, raise p53, reverse EMT phenotype, and increase EOC cells sensitivity to CDDP in vivo and in vitro. High mi R-186 level or low Twist1 expression indicates chemosensitivity, and a longer PFS for EOC patients, and vice versa. These findings clarify the effect and new molecular mechanisms of mi R-186/Twist1 on regulation EMT phenotype and drug resistance in EOC cells, and provide theoretical basis for new diagnostic and therapeutic strategies for ovarian cancer.