PHB1对人肝癌细胞增殖的影响及其作用机制

Effects of prohibitin 1 on cell proliferation of human hepatocellular carcinoma cells and its mechanism

目的 探讨抗增殖蛋白1(prohibitin1,PHB1)对人肝癌细胞增殖的影响及其作用机制。方法 构建PHB1真核表达重组质粒(pEGFP-PHB1)和PHB1靶向siRNA质粒(shRNA-PHB1)转染人肝癌细胞HepG2和SMMC-7721,诱导PHB1在人肝癌细胞中表达上调和下调,采用MTT、流式细胞学、荧光定量PCR和免疫印迹等技术检测人肝癌细胞增殖、细胞周期,以及细胞周期关键调控分子的表达情况。结果 高表达PHB1可阻滞HepG2和SMMC-7721细胞于 G0/G1 期[(67.28±2.94)%比(56.71±2.56)%, t =6.64, P =0.00;(69.48±3.82)%比(60.43±2.59)%, t =4.80, P =0.00],使S期比例下降[(14.74±1.45)%比(24.13±1.92)%, t =9.54, P =0.00;(13.73±1.26)%比(25.50±2.30)%, t =10.99, P = 0.00 ],抑制细胞增殖;周期调控分子p53和p21CIP1mRNA和蛋白质水平显著升高,而Cyclin A2、Cyclin E1和CDK2 mRNA和蛋白质水平显著降低( P <0.01)。低表达PHB1可促使HepG2和SMMC-7721细胞趋于S期[(31.65±2.71)%比(24.68±1.28)%, t =5.69, P =0.00;(31.02±2.49)%比(25.88±2.40)%, t =3.64, P =0.005],促进细胞增殖;p53、p21CIP1、Cyclin A2、Cyclin E1、CDK2 mRNA和蛋白质水平与PHB1高表达时相反( P <0.01)。结论 高表达PHB1可以阻滞人肝癌细胞周期于G0/G1期,进而抑制细胞增殖,其作用机制可能与p53介导的G0/G1期相关细胞周期蛋白有关。

Objective To investigat the effects of prohibitin1 (PHB1) on cell proliferation of human hepatocellular? carcinoma (HCC) cells, and to elucidate the mechanism of PHB1 in the occurrence and development of HCC. Methods HepG2 and SMMC-7721 cells were transfected with enhanced green fluorescent protein plasmid-PHB1 and PHB1-specific short hairpin ribonucleic acid (RNA) to induce the up-regulation and down-regulation of PHB1 expression. The cell proliferation, cell cycle and key regulators of cell cycle were investigated by MTT assay, flow cytometry, real-time polymerase chain reaction and western blot. Results Overexpression of PHB1 arrested the HepG2 and SMMC-7721 cell cycle in G0/G1 phase (67.28%±2.94 vs 56.71%± 2.56, t =6.64, P =0.00;69.48%±3.82 vs 60.43%±2.59, t = 4.80, P =0.00), decreased the proportion of cells in S phase (14.74%±1.45 vs 24.13%±1.92, t =9.54, P =0.00;13.73%±1.26 vs 25.50%± 2.30, t =10.99, P =0.00), and inhibited cell proliferation. Furthermore, as PHB1 overexpressed, the messenger RNA and protein levels of p53 and p21CIP1 were increased, while those of cyclin A2, cyclin E1 and cyclin-dependent kinase 2 were decreased in the HepG2 and SMMC-7721cells ( P <0.01). Conversely, these results were reversed when the expression of PHB1 was inhibited. Conclusion Overexpression of PHB1 arrested the HCC cell cycle in G0/G1 phase, thus inhibit cell proliferation, which might be related to p53-mediated G0/G1 phase-related cell cycle protein.