The Effect Of MACC1Silencing On The Chemosensitivity To Cisplatin In Epithelial Ovarian Cancer Cells Through ERK Pathway

BackgroundAs a kind of malignant tumors,the mortality rate of ovarian cancer ranks the first among gynecology malignant tumor.The trait of ovarian cancer is hidden onset, late diaognosis,early metastasis,quick progress and poor prognosis.At present,the pathogenesis of ovarian cancer is unclear.Bcause of ovarians’locating at the bottom of pelvic,ovarian cancer lacks early distinctive symptoms.When women see a doctor for abdominal distension, about70percent of them are in the later period of cancer. The treatments of late oarian cancer are cytoreductive surgery and chenmotherapy based on platinum. The aim of cytoreductive surgery is remove the tumor tissues at the most limits, but it is inevitable that possible remaining focus is exising.Chemotherapy is essential for ovrian cancer patients. However, some patiens become resistant to chemotherapeutics especially ciasplatin and finaly come to a treatment failure. Chemoresistance is classified into intrinsicly and aqcuied chemoresistance.About15percent to25percent ovarian cancer patiens are intrinsic chemoresistant to chenmotherapy based on platinum, also named primary chenmorisistance.At least80percent patiens gradually become chenmoresistant to cispaltin during chemotherapy, named aqcuied chemoresistance. Chemoresistantce severely restricting living quality and lifetime of ovarian cancer patients.In resent years, researchers are focusing on finding out approaches to improve chemosensitivity.In the year2009, Stein et al found out a new gene in colon cancer,and named it the metastasis-associated in colon cancer-1(MACCl) for the gene was closely associated with the colon cancer.MACC1is a critical regulator of the hepatocyte growth factor (HGF）／C-MET signaling pathway,and enhances the c-met expression on the transcriptional level.MACC1plays a vital role in the invasion, metastasis in colon cancer. Late on, scientists found MACC1expression in many malignant tumors, such as hepatic cell cancer, gallbladder carcinoma, gastric carcinoma, esophagus cancer and breast cancer. Our early reseach found that MACC1mRNA and protein in epithelial oarian cancer was higher than that in belign ovarian tumor and normal ovarian tissues, and then established MACC1specific shRNA to transferred into human ovarian cancer cell OVCAR-3, finding that after transferred with shRNA, the proliferation ability, migration ability and invasion ability of cells in vitro weakened and the apoptosis rate increased. RT-PCR and Western Blot showed that the C-met、p-ERK1/2、cyclinD1、survivin、MMP9、MMP2and VEGFA expreesion of the transffered cells decreased,showing that MACC1may probably participate in the malignant progression of ovarian cancer trough HGF/C-met signaling pathway and downstream MAPK pathway to regulate a series of gene expreesion. However, whether MACC1regulates the chemosensitivity to cisplatin in ovarian cancer has no literature to prove.This paper applied RNAi to inhibit MACC1expression in cisplatin sensitive SKOV3and cisplatin resisitant SKOV3/DDP. RT-PCR was adopted to test MACC1mRNA expression.Western blot was used to measure MACC1, ERK1/2, p-ERK1/2, caspase-3and cleaved caspase-3protein expression. MTT assay was used to measure cell proliferation and sensitivity to cisplatin.Transwell was used to study invasion ability in vitro.This experiment aims at exploring the effect of MACC1silencing on sensitivity to cisplatin of human epithelial ovarian cancer cells,and the ERK pathway expression after MACC1inhibiting,to get the knowledge of whether MACC1affect the chemosensitivity to cisplatin through ERK pathway and provide new reflection on reverse the chemoresistnce. ObjectiveThis paper applied RNAi to inhibit MACC1expression in cisplatin sensitive SKOV3and cisplatin resisitant SKOV3/DDP.The proliferation,apoptosis and chemosensitivity to cisplatin and the molecular mechanism under MACC1influence the above biological behavior was analyzed,The purpose was to provide new reflections on reverse the chemoresistnce clinically. Methods1Transfection of shRNA targeting MACC1mRNA were conducted to ovarian cancer cell SKOV3. MACC1mRNA and protein levels were determined by RT-PCR and Western blot,respectively. ERK1/2and p-ERK1/2protein levels before and after ERK pathway inhibitor PD98059treatment were determined by Western Blot. Chemosensitivity to cisplatin of SKOV3cells was mesuered by MTT assay.2Transfection of shRNA targeting MACC1mRNA were conducted to ovarian cancer cell SKOV3/DDP.Then,MACC1mRNA and protein levels were detected by RT-PCR and Western blot, respectively. ERK1/2、p-ERK1/2、caspase-3and cleaved caspase-3protein levels before and after ERK pathway inhibitor PD98059treatment were determined by Western Blot. Cell proliferation ability and chemosensitivity to cisplatin, apoptosis ratio and migration ability of cells were measured by MTT assay, flow cytometer(FCM) and transwell chamber assays in vitro.3Statistical analysis: the SPSS Statistical package program17.0for all analysis.All data was expressed by mean±standard deviation.One-way ANOVA was used to compare MACC1mRNA and protein,downstream pathway protein,the IC50to cisplatin and apops rate among groups.LSD-t test was used in the means between two groups. Significant level is=0.05. Results1MACC1mRNA and protein levels deceased in SKOV3cells transferred with MACC1shRNA, as well as the expression of p-ERK1/2.The half maximal inhibitory concentration(IC50) of experimental group was lower than that in the other groups. There was a lower expression of p-ERK1/2and more significant decreased IC50in experimental group cells with PD98059added.2MACC1silencing SKOV3/DDP cells showed lower MACC1mRNA and protein levels, lower p-ERK1/2and caspase-3and higher cleaved caspase-3expression than those of parental SKOV3/DDP cells. MACC1silencing cells had lower proliferation and migration ability, higher apoptosis rate and sensitivity to cisplatin than those of parental SKOV3/DDP cells, which could be promoted by PD98059.Conclusion1. The MACC1gene may probably regulate the sensitivity to cisplatin in ovarian cancer cells2. The MACC1gene may probably regulate the proliferation, apoptosis, invasion ability and sensitivity to cisplatin in ovarian cancer cells through ERK pathway.