XAF1基因在肝癌细胞凋亡中的诱导作用

Inducing Effect of XAF1 Gene on Apoptosis in Hepatocellular Carcinoma Cells

背景:X连锁凋亡抑制蛋白(XIAP)与凋亡抑制和肿瘤细胞耐药现象密切相关。目的:研究XIAP相关因子1(XAF1)基因抑制人肝癌细胞株Bel-7404和HepG2增殖和诱导凋亡的作用。方法:以腺病毒Ad5/F35为载体,构建重组腺病毒Ad5/F35-XAF1、对照空病毒Ad5/F35-Null和报告病毒Ad5/F35-增强型绿色荧光蛋白(EGFP),分别以相同感染复数(MOI)感染人肝癌细胞株Bel-7404和HepG2,并设立空白组作为对照。作用48 h后,以荧光显微镜检测Ad5/F35-EGFP的感染效率;分别以逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法检测XAF1 mRNA和蛋白表达;以MTT法检测细胞活力;以Annexin V-FITC/PI双染法和原位末端标记(TUNEL)法检测细胞凋亡率。结果:Ad5/F35-EGFP感染48 h后,几乎所有Bel-7404和HepG2细胞均表达EGFP。Ad5/F35-XAF1感染48 h后,两种肝癌细胞中XAF1 mRNA和蛋白表达均明显增高,细胞活力呈剂量依赖性地降低,细胞凋亡率显著增加。结论:重组腺病毒Ad5/F35-XAF1在人肝癌细胞株Bel-7404和HepG2中的感染效率高,XAF1在不同的肝癌细胞株中恢复表达后,能显著抑制肝癌细胞增殖并促进其凋亡,有可能成为肝癌基因治疗的新靶点。

Background： X-linked inhibitor of apoptosis protein （XIAP） is closely related with apoptosis inhibition and drug resistance of tumor cells. Aims： To investigate the effect of XIAP-associated factor 1 （XAF1） gene on inhibiting proliferation and inducing apoptosis in human hepatocellular carcinoma cell lines Bel-7404 and HepG2. Methods： Recombinant virus Ad5/F35-XAF1, control empty virus Ad5/F35-Null and report virus Ad5/F35-enhanced green fluorescence protein （EGFP） were constructed with the adenovirus vector of Ad5/F35. Human hepatocellular carcinoma cell lines Bel-7404 and HepG2 were infected respectively with these three virus at the same multiplicity of infection （MOI） for 48 hours, and blank group was served as control. The infection efficiency of Ad5/F35-EGFP in Bel-7404 and HepG2 cells was determined by fluorescence microscope. The mRNA and protein expressions of XAF1 were determined by reverse transeriptase polymerase chain reaction （RT-PCR） and Western blotting, respectively. Cell viability was measured by MTT assay, and cell apoptosis was determined by Annexin V-FITC/PI double staining and in situ end-labeling （TUNEL method）. Results： After infection with Ad5/F35-EGFP for 48 hours, almost all Bel-7404 and HepG2 cells expressed EGFP. Fortyeight hours after infection with Ad5/F35-XAF1, the mRNA and protein expressions of XAF1 were significantly increased in these two cell lines. The cell viability was decreased in a dose-dependent manner and cell apoptosis was increased. Conclusions： The recombinant adenovirus Ad5/F35-XAF1 can achieve a high infection efficiency in human hepatocellular carcinoma cell lines Bel-7404 and HepG2. Enhancement of XAF1 expression in hepatocellular carcinoma cell lines can obviously inhibit cell proliferation and induce cell apoptosis. XAF1 is probably a new promising target in gene therapy of hepatocellular carcinoma.