SUMO-1对p53基因诱导HepG2细胞凋亡的增强作用

SUMO-1 Enhances Apoptosis Induced by Wild-Type p53 Plasmid Transfection in HepG2 Cells

目的研究小分子泛素样修饰体1(smallubiquitin likemodifier1,SUMO1)在野生型p53基因诱导HepG2细胞凋亡中的作用。方法用含人野生型p53基因质粒pcDNA3wtp53(pwtp53)、含人双微粒体基因2〔(humandoubleminutegene2(HDM2);鼠同源基因为MDM2〕质粒pCMV HDM1B(pMDM2)、含人SUMO1基因质粒pcDNA3His6SUMO1(pSUMO1)和空质粒pcDNA3分别或同时转染HepG2细胞,获得各转染细胞系,应用Westernblot检测转染后细胞中质粒蛋白的表达及流式细胞技术检测细胞凋亡比例。结果转染pwtp53和pMDM2质粒的HepG2细胞均可见p53及MDM2蛋白条带,同时转染pSUMO1质粒的细胞分别可见被SUMO1修饰的相对分子量较大的p53和MDM2蛋白条带,在未转染任何质粒、仅转染空质粒和pSUMO1质粒的细胞中只检测到少量p53蛋白表达。转染pwtp53及pwtp53+pSUMO1质粒的HepG2细胞凋亡比例分别为(16.79±1.62)%和(18.15±1.36)%,转染pwtp53+pMDM2+pSUMO1质粒的细胞凋亡比例为(14.06±1.84)%,转染pwtp53+pMDM2质粒的细胞凋亡比例则下降至(5.17±1.23)%,与前三者比较差异有显著性意义(P<0.01);其他转染系细胞中的细胞凋亡比例均≤2%,差异无显著性意义。结论SUMO1通过与p53蛋白的结合或翻译后修饰,抑制MDM2等癌基因蛋白对p53蛋白的降解,可增强p53抑癌基因诱导的细胞凋亡。

Objective To investigate whether SUMO-1 enhances the apoptosis induced by wild-type p53 plasmid transfection in HepG2 cells. Methods The HepG2 cells were transfected respectively or simultaneouly with the following expressional plasmids as pcDNA3-wtp53(pwtp53,including human wild-type p53 gene),pCMV-HDM1B(pMDM2,including HDM2 gene, homologous gene as murine double minute gene 2),pcDNA3-His6-SUMO-1(pSUMO-1 ,including small ubiquitin-like modifier-1 gene)and plasmid pcDNA3.The proteins expressed in cells were detected by means of Western blotting and the apoptosis rates of cells were measured by flow cytometry. Results The protein bands of p53 and MDM2 could be seen in cells transfected with pwtp53 and pMDM2. Meanwhile,the relative larger molecular weight bands were also seen in cells transfected with pSUMO-1 which represented the p53 and MDM2 protein modification by SUMO-1. Merely the trace of p53 protein was detected in cells not transfected with any plasmid or only transfected with empty plasmid and pSUMO-1. In cells transfected with pwtp53 and pwtp53+pSUMO-1,the apoptosis rates were (16.79±1.62)% and (18.15±1.36)%. When transfected with pwtp53+pMDM2,the rate decreased to (5.17±1.23)%. The apoptosis rate would come up again to (14.06±1.84)% after transfected with pwtp53+pMDM2+pSUMO-1 and the difference of rates were significant compared to the cells transfected with pwtp53+pMDM2 (P<0.01). The apoptosis rates in other cells were less than 2% and had no significant difference. Conclusion SUMO-1 could increase the apoptosis induced by wild-type p53 plasmid transfection in HepG2 cells through combining to p53 protein or its post-translational modification and inhibiting p53 degradation by MDM2.