P27RF-Rho基因沉默对肝癌Bel7402细胞增殖的影响

目的:构建靶向新分子P27RF-Rho基因的慢病毒RNA干扰载体,研究P27RF-Rho基因对肝癌Bel7402细胞增殖能力的影响。方法:将合成的寡核苷酸片段克隆入RNAi载体,并转染肝癌Bel7402细胞,以沉默P27RF-Rho基因表达。实验分为空白对照Bel7402组、阴性对照Scramble-siRNA组和P27RF-Rho-siRNA组。荧光显微镜观察细胞转染情况;RT-PCR法检测各组Bel7402细胞中P27RF-Rho基因沉默效果;绘制生长曲线,检测转染细胞的增殖情况;平板克隆实验检测细胞的克隆形成能力;RT-PCR法检测细胞增殖能力相关基因P16、Cyclin E、MMP-9和CDK-5 mRNA的表达水平。结果:P27RF-Rho-siRNA组P27RF-Rho基因表达水平明显低于空白对照Bel7402组和阴性对照Scramble-siRNA组(P<0.01)。P27RF-Rho-siRNA组肝癌细胞的生长速度明显低于空白对照Bel7402组和阴性对照Scramble-siRNA组(P<0.05)。与2个对照组比较,P27RF-Rho-siRNA组细胞中CyclinE、MMP-9和CDK-5 mRNA表达水平降低(P<0.01),P16 mRNA表达水平明显升高(P<0.01)。结论:抑制P27RF-Rho基因的表达能够降低肝癌细胞的增殖能力。

Influence of P27RF-Rho gene silencing in proliferation of hepatocelluar carcinoma Bel7402 cells

Objective To construct the RNAi plasmid targeting P27RF-Rho gene, and to explore the effect of P27RF-Rho gene silencing on the proliferation of hepatocelluar carcinoma cells Bel7402. Methods The synthetic oligonucleotide fragment was cloned into RNAi plasmid, and the constructed recombinant plasmid was transfected into the hepatocelluar carcinoma Bel7402 cells for P27RF-Rho gene silencing.This experiment was divided into Bel7402 group, Scramble-siRNA group and P27RF-Rho-siRNA group.The transfection of Bel7402 cells was observed by fluorescence microscope.The gene silencing effect was detected by RT-PCR, and the proliferation of transfected hepatocelluar carcinoma Bel7402 cells was determined by drawing growth curves, and the ability of clone formation was measured by Colony formation assay.The expression levels of P16, CyclinE, MMP-9 and CDK-5 mRNA were detected by RT-PCR. Results The expression level of P27RF-Rho gene in the Bel7402 cells in P27RF-Rho-siRNA group was obviously lower than those in Bel7402 group and Scramble-siRNA group(P<0.01).The growth speed of Bel7402 cells in P27RF-Rho-siRNA group was lower than those in Bel7402 group and Scramble-siRNA group (P<0.05).Compared with Bel7402 group and Scramble-siRNA group, the expression levels of CyclinE, MMP-9 and CDK-5 mRNA in P27RF-Rho-siRNA group were notably decreased (P<0.01), whereas the P16 mRNA expression level was significantly increased (P<0.01). Conclusion The proliferation of hepatocellular carcinoma Bel7402 cells could be controlled by inhibiting the expression of P27RF-Rho gene.