靶向人维吉琳的shRNA真核表达质粒的构建及维吉琳对肝癌细胞周期影响的研究
[Construction of shRNA eucharyotic expression plasmid targeted human VIGILIN and the investigation of VIGILIN's effect on hepatocarcinoma cell cycle].
作者信息
Xie Xiao-yan, Wei Ling, Yang Wen-li, Ge Ya-jun, Qin Yang
机构信息
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
出版信息
Sichuan Da Xue Xue Bao Yi Xue Ban. 2008 Jul;39(4):527-30, 539.
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.
目的
构建针对人维吉琳蛋白（VIGILIN）的短发夹RNA（shRNA）真核表达质粒，并探讨人VIGILIN与肝癌细胞系HepG2细胞周期之间的可能关系。
方法
构建靶向人VIGILIN的shRNA真核表达质粒，并用shRNA表达质粒pSIREN-VIG转染HepG2细胞，然后通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（Western-blot）检测HepG2细胞中VIGILIN mRNA和蛋白的表达，利用流式细胞术（FACS）分析细胞周期的变化。
结果
质粒pSIREN-VIG能有效、特异性地抑制人VIGILIN的表达。转染48小时后，VIGILIN的表达显著降低。由于人VIGILIN表达被敲低，细胞周期受到损害，细胞停滞于G2/M期。各组G2/M期比例分别为：C组（未处理的野生HepG2细胞）2.4%，M组（用转染试剂处理的HepG2细胞）4.9%，G组（用pSIREN-GFP转染的HepG2细胞）6.5%，V组（用pSIREN-VIG转染的HepG2细胞）9.4%。
结论
成功构建了能有效、特异性抑制人VIGILIN表达的shRNA表达质粒。
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