Torin1调节细胞周期抑制人肝癌HepG2细胞增殖的体外研究

Researd on Torin1 Regulation the Cell Cycle and Inhibition Proliferation of Human Liver Hepatocellular Carcinoma Cell Line(HepG2) in Vetro

目的研究Torin1对人肝癌Hep G2细胞生长的影响及其可能机制。方法 CCK8法检测不同浓度(0.1,0.5,1.25,2.5,5)μmol·L-1Torin1对Hep G2细胞48h后细胞增殖的影响;流式细胞仪检测不同浓度(1.25,2.5,5)μmol·L-1Torin1处理前后Hep G2细胞周期的变化,Real time PCR和Western blot法检测不同浓度(1.25,2.5,5)μmol·L-1Torin1处理前后Hep G2细胞细胞周期抑制蛋白P27和细胞周期蛋白A1(cyclin A1)mRNA表达水平和蛋白表达水平的变化。结果Torin1作用于Hep G2细胞后,Hep G2细胞增殖明显呈浓度依赖型抑制,1.25,2.5,5μmol·L-1Torin1作用于Hep G2细胞48h后,细胞周期S期明显增多,P27 mRNA和蛋白表达水平呈浓度依赖性增加,cyclin A1 mRNA和蛋白表达水平呈浓度依赖性减少。结论体外应用Torin1可显著抑制人肝癌细胞Hep G2细胞生长,这可能是通过增加抑制细胞周期蛋白P27表达,减少细胞周期蛋白A1的表达来阻碍细胞周期进程。

OBJECTIVE To investigate in vetro the effect of Torin1 on the proliferation and cell cycle on Hep G2 cell line and explore their molecular mechanism. METHODS The anti-proliferative effect on Hep G2 cells was determined by CCK8 assay. The cell cycle was measured by flow cytometry. The Cyclin A1,P27 expression levels of Hep G2 cells treated with Torin1 were detected in mRNA and protein by Real time PCR and Western blot. RESULTS Torin1 could inhibit cell proliferation on Hep G2 cells,and the inhibitory effects were in dose dependent manners. Cell cycle analysis showed that the S phase of Hep G2 cells treated with Torin1 increased significantly compared with the control group. Real time quantitative PCR and Western blot results showed that Torin1 could down-regulate Cyclin A1 expressions（ P〈0. 05） and up-regulate p27 expression（ P〈0. 05）. CONCLUSION Torin1 has the anti-proliferative effect on Hep G2 cells and induce S phase arrest. These effects might be related with proteins associated with cell cycle closely,such as Cyclin A1 and P27.