【摘要】 目的探讨生长抑制因子(ING1)调控p53信号通路诱导肝癌细胞凋亡的途径。方法通过脂质体转染的方法在体外培养的肝癌细胞株HepG2中过表达ING1基因3种剪接变异体,采用流式细胞仪检测各实验组细胞凋亡率和细胞周期变化,实时荧光定量逆转录聚合酶链式反应(RT-PCR)和Western blot法检测ING1、p53及其下游信号基因bcl-2、bax、p21waf1、mdm2、p14arfmRNA和蛋白表达情况,荧光素酶双标报道基因检测下游p21waf1启动子活性,Western blot检测野生型p53蛋白半衰期变化,使用免疫共沉淀法(IP)观察ING1剪接体与mdm2、p14arf蛋白间的结合情况。结果 HepG2细胞转染过表达ING1不同剪接变异体,pcDNA3-ING1b[(24.36±0.97)%]和pcDNA3-ING1c[(27.33±1.12)%]组细胞凋亡率显著高于空载体组[(8.22%±0.86)%](P<0.01),而pcDNA3-ING1a组细胞凋亡率[(8.90±1.03)%]与空载体组比较差异无统计学意义(P>0.05)。与空载体组比较,pcDNA3-ING1b和pcDNA3-ING1c组均出现G0/G1期阻滞(P<0.05);而pcDNA3-ING1a组各细胞周期的比例与空载体组比较差异无统计学意义(P>0.05)。RT-PCR及Western blot法结果显示过表达ING1b或ING1c使HepG2细胞内源性bax、p21waf1、p14arfmRNA及蛋白表达量明显增高(P<0.01);内源性bcl-2、mdm2 mRNA及蛋白表达量明显降低(P<0.01);过表达ING1a的HepG2细胞bax、bcl-2、p21waf1、mdm2、p14arfmRNA及蛋白表达与空载体组比较差异无统计学意义(P>0.05)。ING1b和ING1c增强p21waf1启动子活性,延长野生型p53蛋白的半衰期,促进p53蛋白乙酰化,并且与p53反馈调节环路基因mdm2和p14arf结合,维持p53蛋白的稳定性和促进p53蛋白的活性。结论 ING1剪接变异体通过与p53负反馈调节基因mdm2竞争性结合p53蛋白,延长野生型p53蛋白半衰期,并促进p53蛋白乙酰化,增强p53蛋白下游基因bax、p21waf1、p14arf表达,抑制bcl-2、mdm2基因表达,促进肝癌细胞凋亡。更多还原

【Abstract】 Objective To explore the effects of inhibitor of growth 1(ING1) gene splicing variants interacting with p53 gene and its signal pathway related genes on inducing hepatoma cells apoptosis.Methods Three recombinant ING1 splicing variants plasmids were transfected into a hepatoma cell line HepG2 with lipofectamine,respectively.Apotosis and cell cycle arrest of HepG2 were analyzed by flow cytometry after transfection.The mRNA and protein expression of ING1,p53,bcl-2,bax,p21waf1,p14arf,mdm2 genes were detected by quantitive reverse transcriptase polymerase chain reaction(RT-PCR) and Western blot.Meanwhile,the luciferase assay was performed to analyze the promoter activity of p21waf1.In addition,Half-life time of wild type p53 protein after transfection was detected by western blot and protein physical binding of ING1 splicing variants with mdm2,p14arfwere explored by using coimmunoprecipitation(IP) method.Results Overexpression of pcDNA3-ING1b or pcDNA3-ING1c variant promoted higher hepatoma cell apotosis [(24.36±0.97) %,(27.33±1.12) %,respectively] and G0/G1arrest compared with the pcDNA3 plain vector control group [(8.22±0.86) % ](P<0.01),while there was no significant difference between ING1a variant [(8.90±1.03) %] and the pcDNA3 plain vector control group(P >0.05).Results of quantitive RT-PCR and western blot showed that,compared with the pcDNA3 plain vector control group,ING1b and ING1c significantly up-regulated the mRNA and protein expression levels of bax,p21waf1,p14arf,but repressed bcl-2 and mdm2 expression(P<0.01).ING1a exerted no obvious influence on the mRNA or protein expression of bax,p21waf1,p14arf,bcl-2 and mdm2(P >0.05).The activity of p21waf1promotor was also strongly enhanced by ING1b or ING1c by using luciferase reporter assay(P<0.01).ING1b and ING1c variant,but not ING1a markedly prolonged the half-life of wild-type p53 and increased p53 acetylation.IP results showed that ING1b and ING1c protein competed with mdm2 protein for binding to p53 protein,and bound to p14arfprotein directly.Conclusion The splicing variants of ING1 enhance hepatoma cell apoptosis and G0/G1cell cycle arrest by activating p53downstream genes bax,p21waf1,p14arf,repressing the expression of bcl-2 and mdm2,competing with mdm2 protein for binding to p53 protein,stabilizing wild-type p53,and increasing the acetylation of p53 protein.更多还原