HBV PS1反式激活蛋白2基因对HepG2细胞增殖和凋亡的影响

Effect of HBV PS1 trans-activator protein-2 gene on proliferation and apoptosis of HepG2 cells

目的沉默乙型肝炎病毒(HBV)PS1反式激活蛋白2基因(PS1TP2)对肝癌细胞HepG2增殖和凋亡的影响。方法荧光定量PCR(RT-PCR)用于检测PS1TP2基因在不同肝细胞系中基础表达水平。将PS1TP2小干扰RNA(siRNA PS1TP2)及对应的阴性对照小干扰RNA(siNC)作为干扰组和对照组分别瞬时转染至HepG2细胞中,培养48 h后,应用CCK-8试剂盒检测两组细胞的增殖活性水平;AnnexinV/7-AAD流式细胞术检测两组细胞凋亡程度;RT-PCR检测两组细胞中PS1TP2、B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)基因mRNA表达水平;Western blot检测两组细胞中Bcl-2、Bax、人腺苷酸活化蛋白激酶(AMPK)、雷帕霉素靶蛋白(m-TOR)蛋白水平并计算Bcl-2与Bax的比例。结果 PS1TP2基因在肝癌细胞HepG2中的表达水平明显高于正常肝细胞L02。干扰组HepG2细胞的增殖速率明显慢于对照组。与对照组相比,干扰组的HepG2细胞中Annexin V+细胞数明显升高( P <0.05),HepG2细胞中Bcl-2 mRNA表达量明显下降( P < 0.05 ),Bax mRNA表达量明显上升( P <0.05);同样在干扰组的HepG2细胞中Bcl-2、m-TOR蛋白水平明显下降( P < 0.05 ),而Bax、AMPK蛋白水平则明显升高( P <0.05)。结论干扰掉PS1TP2基因可经过线粒体途径促进HepG2细胞凋亡,可能是通过活化AMPK-m-TOR途径而抑制其增殖。

Objective To study the effect of HBV PS1 trans-activator protein 2 (PS1TP2) gene silencing on the proliferation and apoptosis of HepG2 cells and its possible mechanism.Methods Fluorescence quantitative PCR (RT-PCR) was used to detect the basal expression of PS1TP2 gene in different liver cell lines.PS1TP2 small interfering RNA (siRNA PS1TP2) and its corresponding negative control small interfering RNA (siNC) as the interference group and the control group,were transiently transfected into HepG2 cells,respectively and cultured for 48 h.CCK-8 kit was used to detect the proliferation activity of the two groups.The apoptosis of the two groups was detected by AnnexinV/7-AAD flow cytometry.The expression of PS1TP2,Bcl-2 and Bax mRNA were detected by RT-PCR.The expression of Bcl-2,Bax,AMP-activated protein kinase(AMPK) and mammalian target of rapamycin (m-TOR) protein were detected by Western Blot and the ratio of Bcl-2 to Bax was calculated.Results The expression of PS1TP2 gene in HepG2 cells was significantly higher than that in normal liver cells L02.Compared with the control group,the proliferation of HepG2 cells in the interfering group significantly decreased ( P <0.05),the number of Annexin V+ cells in HepG2 cells of the interference group significantly increased ( P <0.05),the expression of Bcl-2 mRNA in HepG2 cells of the interference group significantly decreased ( P < 0.05 ),and the expression of Bax mRNA significantly increased ( P <0.05).The expression of Bcl-2 and m-TOR proteins in HepG2 cells of the interference group also decreased significantly ( P <0.05),while the expressions of Bax and AMPK protein increased significantly ( P <0.05).Conclusion PS1TP2 gene interfering can promote the apoptosis of HepG2 cells via mit- ochondrial pathway and may inhibit the proliferation via activiting AMPK-m-TOR pathway.