Ge Ya-Jun, Xie Xiao-Yan, Yang Bo, Chai Xin-Juan, Zhang Ying-Chun, Qin Yang
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2009 Sep;40(5):770-4.
To explore possible relationship among expression of human high density lipoprotein binding protein(VIGILIN), H19 and the insulin-like growth factor 2 (IGF2) mRNA in HepG2 cell cycle and investigate the role of VIGILIN in controlling imprinting genes of H19 and IGF2 mRNA expression.
We investigated time course cell cycle distribution of HepG2 cells by FACS, analyzed VIGILIN, H19 and IGF2 mRNA expression at the indicated times using RT-PCR, RNAi and real-time PCR.
Cell-cycle of HepG2 cells was approximately 20 h. 0 h-9 h and 20 h-28 h, 9 h-20 h and 28 h-39 h were S-phase and G2/M-G1-phase, respectively. Firstly, cells were synchronized by serum-starvation for 24 h. As expected, VIGILIN transcription was up-regulated with expression peaks at 20 h and 60 h after serum stimulating by the addition of 10% fetal calf serum. In parallel, H19 mRNA had a high expression level at 6 h and 43 h, and IGF2 mRNA was also increasing with cell-cycle. The expression profiles of human VIGILIN, H19, and IGF2 mRNA were ascending with cell-cycle. In addition, the knock-down of VIGILIN expression by transfecting HepG2 cells with shRNA expression plasmid pSIREN-VIG inhibited the expression of human VIGILIN, which led to the expression of H19 mRNA decrease by 12.08%, and IGF2 mRNA increase by 30.13%.
The expression of VIGILIN and H19 mRNA was the cell-cycle dependent and had something to do with each other. The results clearly shed light on the roles of VIGILIN in controlling expression of the imprinted H19 and IGF2 genes.
探讨人高密度脂蛋白结合蛋白（vigilin）、H19及胰岛素样生长因子2（IGF2）mRNA在HepG2细胞周期中的表达关系，研究vigilin在调控H19和IGF2 mRNA印记基因表达中的作用。
通过流式细胞术（FACS）研究HepG2细胞的细胞周期时间进程分布，运用逆转录-聚合酶链反应（RT-PCR）、RNA干扰（RNAi）和实时定量聚合酶链反应在指定时间分析vigilin、H19和IGF2 mRNA的表达。
HepG2细胞周期约为20小时。0小时至9小时和20小时至28小时为S期，9小时至20小时和28小时至39小时分别为G2/M期和G1期。首先，通过血清饥饿24小时使细胞同步化。如预期的那样，添加10%胎牛血清刺激血清后，vigilin转录上调，在20小时和60小时出现表达峰值。同时，H19 mRNA在6小时和43小时表达水平较高，IGF2 mRNA也随细胞周期增加。人vigilin、H19和IGF2 mRNA的表达谱随细胞周期上升。此外，用短发夹RNA（shRNA）表达质粒pSIREN-VIG转染HepG2细胞降低vigilin表达，抑制了人vigilin的表达，导致H19 mRNA表达下降12.08%，IGF2 mRNA表达增加30.13%。
vigilin和H19 mRNA的表达依赖于细胞周期且相互关联。这些结果清楚地揭示了vigilin在调控印记基因H19和IGF2表达中的作用。
