The Effection Of ShMRE11on The Drug Resistance And Biological Behavior Of Multidrug-resistant Hepatocellular Carcinoma Cell Line BEL7402/5-FU

Objective:The purpose of this study is to investigate the functions DNA damage repair, chemotherapeutic drug sensitivity,the resistance-associated protein expression and cell biological behavior of resistant hepatic cancer cells BEL7402/5-FU by scilencing MRE11,an important factor about DNA damage repair, and to explore the relationship between MRE11and liver cancer multidrug-resistance.Methods:1.Identification and amplification of shMREll expression vector.2.The human hepatocellular carcinoma BEL7402/5-FU and BEL7402cell culture and the drug resistance identification of BEL7402/5-FU cell by MTT.3.shMRE11and pEGFP-N1was transiently co-transfected into BEL7402/5-FU cells in order to determine the transfection efficiency.4.Evaluation of silence efficiency on mRNA and protein levels by using Real-time PCR and Western blot.5.The protein expression levels of y-H2AX detected by Western blot.6.The cellular DNA synthesis detected by EdU.7.Determination the drug sensitivity to DDP, MMC, ADM,5-FU by using MTT.8.The mRNA and protein expression levels of MRP1detected by Real-time PCR and Western blot.9.The proliferation of cells assayed by MTT.10.The cell cycle determined by Flow Cytometry.11.The cell apoptosis detected by Annexin V-FITC/PI double staining method.Results:1.The results of sequencing showed that the insert sequence of shMRE11plasmid was correct.2.The MTT results showed that the drug resistance index of BEL7402/5-FU to5-FU、ADM、DDP、MMC was11.09、2.05、2.74and2.41respectively.3.The transfection efficiency was47.5%judged by pEGFP-N1.4.Real-time PCR and Western blot results showed the efficiency of RNA interference for MRE11was78.0%on mRNA level and56.1%on protein level.5.Western blot analysis of y-H2AX showed that its expression was higher than control group which the difference was statistically significant(P<0.05).6.EdU detection results showed the DNA synthesis of shMRE11group decreased compared with the control group,which the difference was statistically significant (P<0.05).7.MTT assay results showed that the IC50was significantly lowered of shMRE11group to DDP、MMC、ADM、5-FU compared with the control group,which the difference was statistically significant (P<0.05).8.Real-time PCR and Western blot analysis of MRP1showed that its expression was decreased on both mRNA and protein levels which the differences had statistical significance compared with the control group (P<0.05).9.The cell proliferation determination by MTT showed that the proliferation rate of shMRE11group was slower than the control group(P<0.05).10.The Flow Cytometry results showed that the S-phase fraction of shMRE11group decreased compared with the control group,which the difference was statistically significant (P<0.05).11.AnnexinV-FITC/PI dual staining results showed that the apoptosis index of shMRE11group increased which the difference was statistically significant compared with the control group(P<0.05).Conclusions:Our results display that the shMRE11interference plasmid can effectively inhibit expression of MRE11in BEL7402/5-FU cells, which weaken DNA damage and repair capability, improve the sensitivity for BEL7402/5-FU cells to some anticancer drugs, lower multidrug resistance associated protein MRP1expression, inhibit cell proliferation, reduce S phase and promote apoptosis. To some extent, the silence of MRE11can reduce the drug resistance and the mechanism may also be related to the lower expression of tumor MRP1and the promoting of apoptosis.