肝癌SMMC7721细胞株P27RF-Rho mRNA基因沉默对5-Fu药物敏感性的影响

摘要

Objective:To investigate the influence of P27RF-Rho mRNA gene silencing in the drug sensitivity of 5-fluorouracil(5-Fu)to the liver cancer SMMC cell line,and to provide theoretical basis for the treatment of advanced liver cancer.Methods:The P27RF-Rho RNAi vector was constructed and the P27RF-Rho gene silencing lentivirus were used to infect the SMMC7721 cells.Western blotting method was used to detect the gene silencing effect.The SMMC7721 cells were divided into Scramble-siRNA group, 5-Fu group, P27RF-Rho siRNA group and P27RF-Rho siRNA + 5-Fu group.Western blotting was used to detect the transfection efficiency of RNAi.MTT method was used to detect the cell growth in various groups.Scratching test was used to detect the migration ability of cells in various groups.Transwell experiment were used to detect the invasion ability of cells in various groups.The expressions of P27 and RhoC protein were detected by Western blotting method.Results:P27RF-Rho RNAi lentiviral vector was successfully constructed.The Western blotting results showed that the expression of P27RF-Rho protein in P27RF-Rho siRNA group was decreased compared with 5-Fu group and Scramble-siRNA group(P<0.05).Compared with other three groups, the growth speed of the cells in P27RF-Rho siRNA + 5-Fu group was significantly decreased(P<0.05).The migration ability of the cells in P27RF-Rho siRNA + 5-Fu group was significantly lower than those in other three groups (P<0.01);the average number of cells passing through the Transwell microporous membrane was significantly less than those in other three groups (P<0.01).The Western blotting analysis results showed that the expression level of P27 protein in the cells in P27RF-Rho siRNA + 5-Fu group was significantly higher than those in other three groups(P<0.05);the expression level of RhoC protein was significantly lower than those in other three groups(P<0.05).Conclusion:P27RF-Rho gene silencing can significantly enhance the drug sensitivity of 5-Fu to SMMC7721 cells.%目的:探讨肝癌SMMC7721细胞株P27RF-Rho mRNA基因沉默对5-氟脲嘧啶(5-Fu)药物敏感性的影响,为临床晚期肝癌治疗提供理论依据.方法:构建P27RF-Rho RNAi载体,P27RF-Rho基因沉默慢病毒感染SMMC7721肝癌细胞,Western blotting法检测基因沉默效果.SMMC7721细胞分为Scramble-siRNA阴性对照组、5-Fu组、P27RF-Rho-siRNA组和P27RF-Rho-siRNA+5-Fu组.荧光显微镜检测细胞转染效果,Western blottting法检测RNAi基因沉默效率,MTT法检测各组细胞生长曲线,划痕实验检测细胞迁移能力,Transwell小室检测细胞侵袭能力,Western blotting法检测各组细胞中肿瘤相关蛋白P27及RhoC表达.结果:成功构建P27RF-Rho RNAi慢病毒载体.Western blotting法,P27RF-Rho-siRNA组细胞中P27RF-Rho蛋白表达水平明显低于5-Fu组和Scramble-siRNA组(P<0.05).与其他3组比较,P27RF-Rho-siRNA+5-Fu组细胞生长速度降低(P<0.05);划痕实验中P27RF-Rho-siRNA+5-Fu组细胞迁移能力明显减低(P<0.01);P27RF-Rho-siRNA+5-Fu组穿过Transwell小室微孔滤膜的平均细胞数明显少于其他3组(P<0.01);Western blotting法检测,P27RF-Rho-siRNA+5-Fu组细胞中癌相关蛋白P27表达水平明显高于其他3组(P<0.05),侵袭相关蛋白RhoC表达水平低于其他3组(P<0.05).结论:P27RF-Rho基因沉默能明显增强5-Fu对肝癌SMMC7721细胞的药物敏感性.