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目的 观察慢病毒介导的RNA干扰c-Met基因后肝细胞生长因子(HGF)对结肠癌细胞株SW480侵袭能力的影响.方法 实验分为3组,正常对照组(NC组):正常目的细胞加shRNA阴性对照病毒感染细胞(NC组又分为NC-20和NC-40 2个亚组,分别代表正常目的细胞感染shRNA阴性对照病毒后在Transwell外室中分别加入浓度为20和40 ng/mL的HGF);c-Met基因敲除组(KD组):正常目的细胞加RNA干扰靶点病毒感染细胞(KD组又分为KD-20和KD-40 2个亚组,分别代表正常目的细胞感染目的基因shRNA-c-Met病毒后在Transwell外室中分别加入浓度为20和40 ng/mL的HGF;KD1、KD2、KD3和KD4组分别表示针对目的基因不同的RNA干扰靶点,以挑选其中最有效的干扰靶点);空白对照组(CON组):正常目的细胞加未感染任何病毒的细胞.构建shRNA-c-Met慢病毒表达载体,实时定量PCR (RT-PCR)筛选阳性克隆并测序鉴定;经慢病毒质粒包装后转染SW480细胞,转染48 h后,RT-PCR法检测SW480细胞中c-Met mRNA表达,Western blot法检测SW480细胞中c-Met蛋白表达水平;转染72h后,采用流式细胞仪检测细胞凋亡,Transwell侵袭实验检测细胞的侵袭能力.结果 shRNA-c-Met慢病毒表达载体构建成功;shRNA-c-Met显著下调了SW480细胞中c-MetmRNA的表达,并且RNA干扰靶点病毒感染细胞第4靶点时的基因敲减效率最高(81.4％),为最有效靶点.Western blot检测结果显示,KD组SW480细胞中c-Met蛋白表达明显低于NC组(P=0.015)和CON组(P=0.010),KD组SW480细胞凋亡率明显高于NC组(P＜0.001)和CON组(P＜0.001).细胞转移率在KD组、KD-20组和KD-40组均分别明显低于NC组(P＜0.001)、NC-20组(P=0.015)及NC-40组(P=0.017);与NC组比较,NC-20组和NC-40组的细胞转移率明显升高(P＜0.001、P＜0.001),且NC-40组的细胞转移率明显高于NC-20组(P=0.005);同样,与KD组比较,KD-20组和KD-40组的细胞转移率均明显升高(P＜0.001、P＜0.001),KD-40组的细胞转移率明显高于KD-20组(P=0.014).结论 以c-Met为靶点的RNA干扰能明显下调结肠癌细胞株SW480中c-Met的表达,c-Met的表达下调能明显增加细胞凋亡且能降低HGF对SW480细胞侵袭能力的影响.“,”Objective To investigate effect ofhepatocyte growth factor (HGF) afterlentivirus-mediatedRNA interference (RNAi) targeting c-Met on invasion of colonic carcinoma cell line SW480.Methods The experiment was assigned into 3 groups:NC group,the normal cells were infected by the shRNA negative control virus (the NC-20 and NC-40 represented the negative group which were added 20 ng/mL and 40 ng/mL respectively HGF after being infected);KD group,the normal cells were infected by the shRNA-c-Met target virus (the KD-20 and KD-40 represented the interfered group which were added 20 ng/mL and 40 ng/mL HGF respectively after being infected;KD 1,KD2,KD3,and KD4 represented the different RNAi targets for the purpose gene);CON group,the normal cells were not infected by any virus.The lentiviral vector shRNA-c-Met was constructed and verified by polymerase chain reaction (PCR) and DNA sequencing.The SW480 cells were infected with the shRNA-c-Met after packed with lentivirus plasmid.Fourty-eight hours transfection later,the c-Met mRNA of the transfected SW480 cell was detected by real time PCR and the c-Met protein was examined by Western blot.Seventy-two hours after transfection,the cell apoptosis was detected by flow cytometry and the invasions in the different cells with stable transfection were detected by Transwell test.Results The RNAi sequence targeting c-Met gene was successfully inserted into the lentiviral vector.The shRNA-c-Met transfection resulted in an obviously reduced expression of c-Met mRNA in the SW480 cells.The efficency of gene knock down of the KD4 (the cells with No.4 target spot knocked down) was 81.4％.The shRNA-c-Met tansfection resulted in an obviously reduced expression of c-Met protein in the SW480 cells.After transfection,the apoptosis rate of the KD group was significantly higher than that in the NC group (P＜0.001) or the CON group (P＜0.001).The invasion ratios in the NC group,NC-20 group,and NC-40 group were significantly higher than those in the KD group (P＜0.001),KD-20 group (P=0.015),and KD-40 group (P=0.017),respectively;which in the NC-20 group and NC-40 group were increased as compared with the NC group (P＜0.001,P＜0.001),and in the NC-40 group was increased as compared with the NC-20 group (P=0.005).The invasion ratios in the KD-20 group and KD-40 group were increased as compared with the KD group (P＜0.001,P＜0.001),and in the KD-40 group was increased as compared with the KD-20 group (P=0.014).Conclusion Lentivirus-mediated RNAi targeting c-Met could effectively suppress expression of c-Met in SW480 cells and could reduce invasion of HGF on SW480 cells with knocked down c-Met.