PEG10基因siRNA真核表达载体对HepG2细胞周期的影响

目的观察针对遗传印记基因PEG10的siRNA真核表达载体对人肝癌HepG2细胞周期及细胞周期调控蛋白表达水平的影响。方法以脂质体法将psiRNA-PEG10真核表达载体转染HepG2细胞，RT-PCR检测PEG10mRNA的表达，流式细胞仪观察细胞周期分布的变化；RT-PCR和Western Blotting法分别检测细胞周期调节蛋白D1（Cyclin D1）和P27mRNA和蛋白水平的表达变化。结果RT-PCR结果显示，转染psiRNA-PEG10的HepG2细胞中PEG10mRNA的表达明显降低；流式细胞仪检测发现，与未转染组和空载体组相比，转染psiRNA-PEG10后处于G0／G1期的HepG2细胞百分比明显升高（P〈0．01），而G2／M期的细胞百分比明显降低（P〈0．01）；在mRNA和蛋白水平上，转染psiRNA-PEG10的细胞Cyclin D1表达明显降低；而P27表达明显升高（P〈0．01）。结论PEG10可通过影响细胞周期调控蛋白CyclinD1和P27表达，干扰肿瘤细胞的细胞周期发挥抗瘤作用，针对PEG10的siRNA真核表达载体有望成为研究肝癌治疗的有力工具。

Effects of siRNA enkaryotic expression vectors targeting PEG10 gene on cell cycle in HepG2 cells

Objective To investigate the effects of siRNA eukaryotic expression vectors targeting PEG10 on HepG2 cell cycle and cell-cycle regulatory proteins. Methods The psiRNA-PEG10 plasmid was transiently transfected into HepG2 cell with Lipofectamine 2000. The expression of PEG10 in HepG2 was detected by RT-PCR. The effect of PEG10 on the cell cycle was observed by flow cytometry. The mRNA and protein expression of Cyclin D1and p27 were detected by RT-PCR and Western Blotting respectively. Results There was a depressed expression of PEG10 in the cells transfected with psiRNA-PEG10. Upon flow cytometry, there were more cells in G0/G1 phase and fewer cells in G2/M phase in psiRNA-PEG10-transfected cells. The mRNA and protein expression of Cyclin D1 decreased in the psiRNA-PEG10-transfected cells, while p27 expression increased (P<0.01). Conclusion The result indicated that PEG10 might play an important role in the pathogenesis of HCC by inhibiting the cell cycle, which was related to the decreased Cyclin D1 and increased p27 expression. SiRNA eukaryotic expression vector targeting PEG10 may become a promising-treatment strategy of HCC.