人VIGILIN的shRNA真核表达质粒的构建及其对肝癌细胞周期影响的初探

目的构建靶向人高密度脂蛋白结合蛋白(VIGILIN)的shRNA真核表达载体,并初步探索VIGILIN与人肝癌细胞系HepG2细胞周期是否有关联。方法构建人VIGILIN的shRNA重组质粒,并将重组质粒pSIREN-VIG转染HepG2细胞,采用RT-PCR和Western-blot检测HepG2细胞VIGILIN mRNA和蛋白的表达,并用流式细胞术检测细胞周期是否有所改变。结果1HepG2细胞转染pSIREN-VIG后,VIGILIN的表达被特异、有效地抑制。转染48h后,VIGILIN的表达从mRNA和蛋白的水平都有明显的减少;2VIGILIN表达抑制后,G2/M期细胞所占比例增加:相对于三个对照组(未转染组2.4%,脂质体转染组4.9%和转染pSIREN-GFP组6.5%),实验组(转染pSIREN-VIG组)G2/M期细胞增加到9.4%。结论我们成功的构建了能特异、有效的抑制人VIGILIN表达的shRNA表达质粒,人VIGILIN能够影响到细胞周期的正常运行,导致G2/M期阻滞。

Construction of shRNA Eucharyotic Expression Plasmid Targeted Human VIGILIN and the Investigation of VIGILIN's Effect on Hepatocarcinoma Cell Cycle

Objective To construct a shRNA eucharyotic expression plasmid against human VIGILIN and explore possible relation between human VIGILIN and HepG2 cell cycle.Methods We constructed the shRNA eucharyotic expression plasmid targeted human VIGILIN,and transfected HepG2 cells with shRNA expression plasmid pSIREN-VIG,then determined the expression of VIGILIN mRNA and protein in HepG2 cells by RT-PCR and Western-blot,analysed alteration of cell cycle using FACS.Results The plasmid pSIREN-VIG can effectively and specifically inhibit the expression of human VIGILIN.After transfection 48 hours,the expression of VIGILIN was significantly decreased.Due to knockdown of human VIGILIN,cell cycle is impaired and cells are arrested in G2/M phase.The proportion of G2/M phase of all groups were listed as:C group(untreated wild HepG2 cells)2.4%,M group(HepG2 cells treated with transfection reagent)4.9%,G group(HepG2 cells transfected with pSIREN-GFP)6.5% and V group(HepG2 cells transfected with pSIREN-VIG)9.4%.Conclusion We have successfully constructed a shRNA expression plasmid which could effectively and specifically inhibit the expression of human VIGILIN.