HBx基因下调miR-192对人肝癌细胞株HepG2周期的影响

Effects of Down-regulation of miR-192 by HBx Gene on Cell Cycle of HumanHepatocellular Carcinoma Cell Line HepG2

目的探讨HBx基因通过调节miR-192的表达影响人肝癌细胞株HepG2周期进展的机制。方法流式细胞仪分析以下3组细胞的周期变化:HepG2/HBx细胞(HepG2细胞稳定转染HBx基因)、HepG2/pcDNA3.1细胞(HepG2细胞稳定转染空载体pcDNA3.1)以及HepG2细胞。3组细胞中miR-192的表达采用Taqman探针荧光定量PCR法检测。流式细胞术观察转染miR-192后HepG2细胞的周期分布变化,SYBR Green荧光定量PCR和Westernblot分别检测miR-192对HepG2细胞p53、CDKN1A mRNA和蛋白表达的影响。结果 3组细胞中,HepG2/HBx细胞G0/G1期细胞比例明显降低[(52.78±4.08)%vs(67.37±4.87)%,(65.08±5.15)%],S期和G2/M期比例明显升高[S期:(25.22±1.84)%vs(19.78±1.26)%,(18.84±1.68)%;G2/M期:(22.00±2.07)%vs(12.85±1.29)%,(16.08±1.44)%]。HepG2/HBx细胞miR-192表达显著下调[(49.1±5.9)%vs(98.0±8.9)%,(100.0±9.1)%]。转染miR-192引起HepG2细胞G0/G1期和G2/M期阻滞,同时p53、CDKN1A mRNA(p53:1.68±0.12 vs 0.90±0.06;CD-KN1A:2.36±0.12 vs 1.05±0.06)和蛋白(p53:3.07倍;CDKN1A:2.82倍)的表达水平亦显著上升。结论 miR-192通过促进p53和CDKN1A表达引起HepG2细胞周期阻滞,而HBx通过下调miR-192加速HepG2细胞周期进程。

Objective To investigate the mechanism of HBx gene on cell cycle of human hepatocellular carcinoma cell line HepG2 through miR-192 regulation.Methods Flow cytometry was used to analyze the cell cycle of these three cell lines：HepG2/HBx cells（HepG2 cells stably transfected with HBx）,HepG2/pcDNA3.1 cells（HepG2 cells stably transfected with pcDNA3.1）and HepG2 cells.MiR-192 expression of these cells was detected by Taqman fluorescence quantitative PCR.Following transfection with miR-192,cell cycle distribution of HepG2 cells was tested by flow cytometry,and SYBR Green quantitative PCR and Western blot were used to examine the expression of p53 and CDKN1A mRNA and protein.Results The proportion of HepG2/HBx cells was decreased significantly in G0/G1 phase [（52.78±4.08）% vs（67.37±4.87）%,（65.08±5.15）%],but increased remarkably in S phase and G2/M phase [S phase：（25.22±1.84）% vs（19.78±1.26）%,（18.84±1.68）%;G2/M phase：（22.00±2.07）% vs（12.85±1.29）%,（16.08±1.44）%].The expression of miR-192 was down-regulated in HepG2/HBx cells [（49.1±5.9）% vs（98.0±8.9）%,（100.0±9.1）%].Transfection of miR-192 into HepG2 cells caused cell cycle arrest at G0/G1 and G2/M phases.Meanwhile the mRNA（p53：1.68±0.12 vs 0.90±0.06;CDKN1A：2.36±0.12 vs 1.05±0.06）and protein（p53：3.07-fold;CDKN1A：2.82-fold）expression levels of p53 and CDKN1A genes were dramatically increased.Conclusion miR-192 could induce cell cycle arrest by augmentation of p53 and CDKN1A levels in HepG2 cells and HBx could down-regulate the expression of miR-192 to facilitate cell cycle progression.