c-Met基因慢病毒载体的构建及其配体肝细胞生长因子对SW480细胞侵袭能力的影响

c-Met expression after lentivirus-mediated RNA interference and effect of hepatocyte growth factor on invasion of colonic carcinoma cells SW480 with knocked down c-Met

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sh RNA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sh RNA阴性对照病毒后在Transwell外室中分别加入浓度为20和40 ng/m L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sh RNA-c-Met病毒后在Transwell外室中分别加入浓度为20和40 ng/m L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sh RNA-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 m RNA表达,Western blot法检测SW480细胞中c-Met蛋白表达水平;转染72 h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 sh RNA-c-Met慢病毒表达载体构建成功;sh RNA-c-Met显著下调了SW480细胞中c-Met m RNA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） after lentivirus-mediated RNA 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.