RNA干扰真核翻译延长因子1A1抑制肝癌Hep3B细胞的增殖

The inhibition of proliferation of hepatocellular carcinoma Hep3B cells induced by RNA interference targeting eEF1A1

目的 :探讨RNA干扰真核翻译延长因子1A1(eukaryotic translation elongation factor 1A1,e EF1A1)的表达对肝癌Hep3B细胞增殖能力的影响及其可能的作用机制。方法 :构建靶向e EF1A1的短发夹RNA(short hairpin RNA,sh RNA)重组载体p GPU6/GFP/Neo-e EF1A1-sh RNA,并将其转染至肝癌Hep3B细胞中,随后应用实时荧光定量PCR和蛋白质印迹法分别检测e EF1A1 m RNA和蛋白的表达,CCK-8(cell counting kit 8)法检测细胞的增殖能力,平板克隆形成实验检测细胞的克隆形成能力,FCM法检测细胞周期的变化,蛋白质印迹法检测细胞周期蛋白D1(cyclin D1)和周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)的表达。结果 :成功构建重组载体p GPU6/GFP/Neo-e EF1A1-sh RNA,并获得稳定低表达e EF1A1的肝癌Hep3B细胞。p GPU6/GFP/Neo-e EF1A1-sh RNA转染组Hep3B细胞中e EF1A1 m RNA和蛋白的表达水平明显低于阴性对照组(转染阴性对照载体p GPU6/GFP/Neo-NC的Hep3B细胞)和空白对照组(未转染的Hep3B细胞)(P<0.01,P<0.05)。与阴性对照组比较,p GPU6/GFP/Neo-e EF1A1-sh RNA转染组Hep3B细胞的体外增殖能力和克隆形成能力明显下降(P<0.05,P<0.01),G1期细胞所占比例明显上升(P<0.01),S期细胞所占比例明显下降(P<0.05),cyclin D1和CDK4蛋白的表达水平明显下调(P<0.05)。结论 :下调e EF1A1的表达可以抑制肝癌Hep3B细胞的增殖能力。

Objective：To investigate the effects of RNA interference targeting eukaryotic translation elongation factor 1A1（e EF1A1） expression on the proliferation of hepatocellular carcinoma Hep3 B cells,and to explore its possible mechanism.Methods：A recombinant vector p GPU6/GFP/Neo-e EF1A1-sh RNA containing short hairpin RNA（sh RNA） targeting e EF 1A 1 was constructed,and then it was transfected into the Hep3 B cells.After tansfection with p GPU6/GFP/Neo-e EF1A1-sh RNA,the expressions of e EF1A1 m RNA and protein were examined by real time fl uorescence quantitative PCR and Western blotting,respectively,the cellular growth ability was examined by cell counting kit 8（CCK-8） assay,the colony formation ability was detected by colony formation assay,and the cell cycle distribution of Hep3 B cells was detected by fl ow cytometry（FCM）.The expression levels of cyclin D1 and cyclin-dependent kinase 4（CDK4） proteins in Hep3 B cells were examined by Western blotting.Results：The recombinant vector p GPU6/GFP/Neo-e EF1A1-sh RNA was successfully constructed,and the Hep3 B cells with stable expession of e EF1A1 were established.The expression levels of e EF1A1 m RNA and protein in Hep3 B cells after transfection with p GPU6/GFP/Neoe EF1A1-sh RNA were lower than those in the negative control cells（Hep3B cells transfected with a negative control vector p GPU6/GFP/Neo-NC） and the blank control cells（Hep3B cells without any transfection）（P 〈 0.01,P 〈 0.05）.As compared with the negative control,the cellular growth ability and the colony formation ability of Hep3 B cells after tranfection with p GPU6/GFP/Neo-e EF1A1-sh RNA were obviously decreased（P 〈 0.05,P 〈 0.01）; the percentage of the cells in G1 phase was increased（P 〈 0.01）,and which in S-phase was decreased（P 〈 0.05）; the expression levels of cyclin D1 and CDK4 proteins were down-regulated（both P 〈 0.05）.Conclusion：Down-regulation of e EF1A1 expression can inhibit the proliferation of Hep3 B cells.