乙型肝炎病毒X基因下调miR-192对人肝癌细胞株HepG2凋亡的影响

目的 探讨乙型肝炎病毒X基因(HBx)通过调节人肝癌细胞株HepG2中miR-192的表达而抑制其凋亡的机制.方法 设立3个细胞组:稳定转染HBx基因的HepG2细胞(HepG2/HBx),稳定转染空载体pcDNA3.1的HepG2细胞(HepG2/pcDNA3.1)以及未作转染的HepG2细胞.用流式细胞术分析3个细胞组的凋亡率差异,用Taqman探针荧光定量PCR检测3组细胞中miR-192的表达水平.转染miR-192后,用流式细胞术检测HepG2细胞凋亡率的变化,同时用SYBR Green荧光定量PCR和Western blot检测细胞中p53、PUMA表达的变化.计量资料均数的比较用单因素方差分析.结果 HepG2/HBx细胞的凋亡率为2.37％±0.35％,较HepG2/pcDNA3.1、HepG2细胞(11.46％±0.69％、12.50％±0.66％)明显降低(F=171.722,P＜0.01).miR-192表达在HepG2/HBx细胞中为49.1％±5.9％,较HepG2/pcDNA3.1、HepG2细胞(98.0％±8.9％,100％)也明显下调(F=14.319,P＜ 0.05).转染miR-192后HepG2细胞的凋亡率(15.74％±1.17％)较转染相应阴性对照的HepG2细胞的凋亡率(10.74％±1.15％)显著升高(F=18.415,P＜0.05),同时,p53、PUMA基因在mRNA (953:1.68±0.12比0.90±0.09,F=43.115,P＜0.05 ; PUMA:1.66±0.10比0.98±0.06,F=22.541,P＜0.05)和蛋白质水平(p53:3.07比1,PUMA:2.13比1)的表达均显著上升.结论 miR-192促进HepG2细胞凋亡,HBx通过下调miR-192抑制HepG2细胞凋亡.

HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2

Objective To explore the mechanism by which H-BV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA.Methods Three cell lines were prepared:HepG2 cells stably transfected with HBx (HepG2/HBx),HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells.Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression.After HepG2 cells was transfected with miR-192,the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot,respectively.Results Compared with HepG2/pcDNA3.1 cells (11.46％ ± 0.69％) and HepG2 cells (12.5％ ± 0.66％),the apoptosis rate of H epG2/HBx cells (2.37％ ± 0.35％) was significantly reduced ( F =171.722,P ＜ 0.01 ).The level of miR192 was 49.1％ ± 5.9％ in HepG2 cells,which was dramatically down-regulated (F=14.319,P =0.019) as compared to the other two groups (HepG2/pcDNA3.1:98.0％ ± 8.9％; HepG2:100％).Compared with HepG2 cells transfected with miR-NC (10.74％ ± 1.15％),transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74％ ±1.17％) ( F =18.415,P =0.013) and higher p53 and PUMA expressions at mRNA (p53:1.68 ± 0.12 vs 0.90 ± 0.09,F =43.115,P =0.003,PUMA:1.66 ± 0.10 vs 0.98 ± 0.06,F =22.541,P =0.009)and protein (p53:3.07 vs 1,PUMA:2.13 vs 1 ) levels.Conclusion HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR- 192 which induces apoptosis of HepG2 cells.