Tet-on基因表达系统调控HIF-1α表达对肝癌细胞增殖的影响

Effects of HIF-1α expression regulated by Tet-on gone expression system on hepatoma cell proliferation and cell cycle in vitro

目的 探讨HIF-1α表达对肝癌细胞增殖和细胞周期的影响.方法 利用Tet-on基因表达系统调控肝癌细胞系HepG2中HIF-1α的表达,检测细胞周期和细胞增殖的变化.结果 酶切和DNA测序证实Tet-on基因表达系统反应质粒PTRE-HIF-1α构建成功.获得了受强力霉素调控、稳定表达HIF-1α的肝癌细胞.随着强力霉素浓度增加,hiF-1α基因表达增加,HIF-1α在体外明显增加HepG2细胞增殖活性;G0/G1期细胞指数减少,细胞增殖指数增多.RT-PCR检测结果表明,随着HIF-1α基因表达增加,Cyclin A mRNA表达增加（P＜0.001）,Cyclin D1和Cyclin E mRNA表达水平无明显变化（P＞0.05）.结论 HIF-1α基因在体外可以通过HIF-1α表达水平的增加而促进肝癌细胞增殖活性,通过CyclinA表达增加缩短了肝癌细胞增殖周期.

Objective To investigate the effects of HIF-1α expression regulated by Tet-on gene expression system on cell proliferation and cell cycle of hepatoma cells in vitro. Methods The change of human hepatocellular carcinoma cell lines HepG2 cell cycle and cell proliferation was measured after HIF-1 α expression of HepG2 in vitro was regulated by Tet-on expression system. Results Amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing, and pTRE-HIF-1α obtained by edonuclease digestion,capable of expression in HepG2 Tet-on cells. After being incubated under different concentrations of doxycycline for 48 h, MTT assays showed that up-regulation of HIF-1α expression increased HepG2 cell proliferation activities. The cell index of S and G2/M phase was significantly higher and that of G0/G1 phase reduced with the increasing concentrations of doxycycline. The mRNA expression of Cyclin A increased with the increasing concentrations of doxycycline （ P 〈 0. 001 ）, CyclinD1 and CyclinE did not change （ P 〉0. 05）. Conclusion HIF-1 α gene promotes cell proliferation and cell cycle of hcpatoma cells in vitro, and this effects increased with the increasing of HIF-1α expression possibly through influencing the expression of CyclinA.