肝癌细胞中RhoA小干扰RNA载体的构建与鉴定

Construction and identification of RhoA specific siRNA expression vector in hepatocellular carcinoma cell

目的构建靶向 RhoA 基因的 RNA 干扰(RNAi)载体。方法根据人 RhoA 基因序列设计两条短双链,人工合成后,连接到真核表达载体 Pgenesil-1中,构成 Pgenesil-1-siRhoA1、Pgene-sil-1-siRhoA2两个重组质粒,转入人肝癌细胞(HEPG2)中,Western blot 方法检测 RhoA-siRNA 对 RhoA表达的抑制效果,确定一个重组质粒进行下一步研究。结果两质粒都有沉默 RhoA 基因的作用,Pgen-esil-1-siRhoA1重组质粒作用更强烈一些,使用 Pgenesil-1-siRhoA1质粒进行下一步研究。结论 Pgen-esil-1-siRhoA 重组质粒的成功构建,为研究 RhoA 与肝细胞癌侵袭转移的关系提供实验模型。

Objective To construct RhoA siRNA plasmid expression vector.Methods According to the computer aided design,RhoA-specific siRNA gene was synthesized and cloned into the RNAi-Ready Pgenesil-1 Vector.The constructed RhoA-RNAi plasmid were transfected into human HEPG2 cell.Western blot was used to detect the effect of RhoA-RNAi plasmid.Results The recombinant was cloned and the se- quence was obtained.RhoA-RNAi plasmid can down-regulate the expression of RhoA in human hepatocel- lular carcinoma cell line HEPG2.Conclusion Successfully cloning the recombinant makes it possible for searching new mechanism of RhoA in hepatocellular carcinoma.