基因沉默RhoC联合雷帕霉素对肝癌细胞侵袭和转移影响的实验研究

Combination of RhoC-siRNA with Rapamycin influence on invasion and metastasis of hepatocellular carcinoma cell BEL7402

目的:通过基因沉默RhoC联合雷帕霉素阻断mTOR通路对肝癌细胞侵袭和转移影响的实验研究,证明二者联合能显著抑制肝癌侵袭、迁移,寻找治疗肝癌的新分子靶点。方法:RhoC-siRNA质粒转染BEL7402细胞,荧光显微镜检测转染效率;Western blot方法鉴定RhoC的基因沉默效果。实验分为6组:空白对照BEL7402组、阴性对照Scramble-siRNA组、阴性对照Scramble-siRNA+Rapa组、Rapa组、RhoC-siRNA组、RhoC-siRNA+Rapa组。采用半定量RT-PCR法检测侵袭相关因子MMP2、MMP9、TIMP1、TIMP2、P27RF-Rho以及凋亡相关因子Survivin表达;免疫细胞化学法检测MMP2、MMP9的表达;采用划痕实验,细胞黏附实验,Transwell小室检测细胞的侵袭转移。结果:Scramble-siRNA+Rapa组、Rapa组、RhoC-siRNA组MMP2、MMP9、TIMP1、TIMP2、p27RF-Rho、Survivin和空白对照BEL7402组、阴性对照Scramble-siRNA组比较表达降低(P<0.05),RhoC-siRNA联合Rapa组的表达显著降低(P<0.01);Scramble-siRNA+Rapa组、Rapa组、RhoC-siRNA组抑制肝癌细胞侵袭和迁移(P<0.05),而RhoC-siRNA联合Rapa组显著抑制肝癌细胞的侵袭迁移(P<0.01)。结论:RhoC-siRNA联合雷帕霉素能显著抑制肝癌细胞的迁移、侵袭,是治疗肝癌的良好分子靶点。

Objective：To prove the correlation between the function of RhoC signaling pathway and the mTOR one, and find the new molecule-remedy target through the research of RhoC-siRNA combined with Rapamycin（Rapa） influencing on the invasion and metastasis of HCC. Methods： The plasmid RhoC-siRNA conducted was used to transfected into BEL7402 ceils, and the transfection rate was detected by fluorescence microscope. Western Blot analysis was identified for the effect of transfection. This experiment was di- vided into six groups：blank control group, negative plasmid group, negative plasmid group with Rapa, RhoC-siRNA group, Rapa group, RhoC-siRNA combined with Rapa group. The factor MMP2, MMP9, TIMP1, TIMP2, P27RF-Rho and the anti-apoptosis factor survivin were determined by PCR. And MMP2, MMP9 were examined by immunocytochemical method. The invasion and metastasis of ceils were detected by wound assay, cell adhesion experiment, Transwell invasion assay. Results： The expression of Scramble-siRNA with Rapa group, Rapa group and RhoC-siRNA group compared to control group and Scramble-siRNA group were decreased （P 〈 0.05 ）, the ex- pression of RhoC-siRNA combined with Rapa group were significantly reducing（P 〈 0.01 ）, the Scramble-siRNA with Rapa group, Ra- pa group and RhoC-siRNA group could prevent the invasion and metastasis of HCC （ P 〈 0.05 ）. The RhoC-siRNA with Rapa group re- strained the invasion and metastasis dramatically（P 〈 0.01 ）. Conclusion： The combination of RhoC-siRNA with Rapa could restrict evidently hepatocelluar carcinoma cell＇ s invation and migration, and this combination is a good target that curing the liver cancer.