MiR-30c通过靶向KRAS抑制肝癌细胞系生长侵袭能力的体外研究

Mi R-30c inhibits proliferative and invasive abilities of human hepatocellular carcinoma cell line in vitro through KRAS signaling pathway

目的探讨Mi R-30c对肝癌细胞生长和侵袭的影响。方法采用脂质体Lipofectamine 2000将Mi R-30c模拟物组和Mi R-30c模拟物组阴性对照转染至肝癌细胞Huh7,q RT-PCR检测转染效果,细胞免疫荧光、Western blot检测转染后目的蛋白的表达,Transwell、划痕实验检测细胞迁移侵袭能力、MTT法检测细胞增殖活性,流式细胞术检测细胞周期分布的改变。结果成功构建稳定过表达Mi R-30c的肝癌细胞亚系Huh7-Mi R30c,荧光定量PCR结果显示Mi R-30c表达量明显上调,过表达Mi R-30c后Huh7细胞的侵袭和增殖能力均受到明显抑制;抑制Mi R-30c能明显抑制KRAS蛋白和减低p-ERK蛋白表达水平,对ERK蛋白水平无明显影响。结论抑制Mi R-30c表达可能通过调控RAS/MAPK/ERK通路抑制肝癌细胞Huh7增殖和侵袭。

[Objective]To investigate the effects of miR-30 c on the growth and invasion of hepatocellular carcinoma cells.[Methods]Mi R-30 c mimics and miR-30 c mimic negative control were transfected into human hepatocellular carcinoma cell line Huh7 by Lipofectamine 2000. The transfection efficiency was detected by real-time PCR. The target protein was detected by Western blot. Finally, scratch test, transwell cell invasion, MTT and flow cytometry were used to detect the migratory, invasive and proliferative activities of the tumor cells.[Results]A cell subclone stably overexpressing miR-30 c was obtained and verified by real-time quantitative PCR. Mi R-30 c overexpression significantly inhibited the invasion and proliferation of Huh7 cells. Inhibition of miR-30 c did not affect the expression of total ERK, whereas the phosphorylated ERK level was markedly down-regulated and the KRAS protein expression was down-regulated.[Conclusions]Overexpression of miR-30 c could inhibit the migration and proliferation of human hepatocellular carcinoma cell line Huh7 via the RAS/MAPK/ERK pathway.