RNA干扰GPC3基因表达影响肝癌细胞Huh-7凋亡的机制研究

Inhibition of GPC3 expression via RNA interference influences the apoptosis of a human hepatocellular carcinoma cell line Huh-7

目的观察RNA干扰GPC3基因表达对人肝癌细胞Huh-7凋亡的影响,并探讨其初步机制。方法设计并合成靶向GPC3的特异性siRNA片段,通过脂质体转染法瞬时转染肝癌细胞Huh-7,48h后验证siRNA的干扰效率;Annexin V/PI染色法观察GPC3基因沉默对细胞凋亡的影响;通过Western blot和定量PCR来检测caspase3的蛋白水平和核酸水平表达。结果设计合成的GPC3siRNA转染后,能够有效抑制Huh-7细胞中GPC3的表达;流式细胞仪检测结果显示,GPC3干扰组细胞在转染后48、72h的凋亡率显著高于空白对照组(P<0.01),96h后两组凋亡率差异无统计学意义(P>0.05)。GPC3干扰组的caspase3在转染后48、72h的表达高于未转染组,96h后两组间caspase3水平差异无统计学意义(P>0.05)。结论 siRNA静默GPC3基因能促进肝癌细胞凋亡,其机理可能与上调caspase3有关。进而推测,GPC3基因可能成为肝癌靶向治疗的一个新靶点。

Objective To investigate the effect of RNA knockdown of the GPC3 gene on the apoptosis of hepatocellular carcinoma Huh-7 cells.Methods siRNA targeting GPC3 was designed and synthesized,and then transfected into the Huh-7 cells via Lipofectamine 2000 mediation.The cell apoptotic rate was analyzed by using flow cytometry.Expression of capase3 was detected by RT-PCR and western blot.Results GPC3-targeted siRNA could down-regulate the GPC3 expression of Huh-7.Compared with control group,cell apoptosis rates were significantly increased in siRNA interference group at 48 h and 72 h after transfection（P0.05）.Expression of caspase-3 in siRNA interference group was significant higher than that of control group at 48 h and 72 h after transfection（P0.05）.Conclusion Down-regulation of GPC3 gene can induce apoptosis of HCC Huh-7 cells.It could be regarded as a novel target for clinical diagnosis and gene therapy for HCC.