生长抑制因子1剪接变异体调控p53信号通路诱导肝癌细胞凋亡  

Inhibitor of Growth 1 Splicing Variants Induce Hepatoma Cells Apoptosis Through Interacting with p53 and Its Signal Pathway Related Genes

作　　者：祝峙[1] 冯真[1] 张晶[1] 史敏[1] 朱焱[1] 郑建明[1] 

机构地区：[1]第二军医大学附属长海医院病理科,上海200433

出　　处：《肿瘤基础与临床》2013年第5期369-375,共7页journal of basic and clinical oncology

基　　金：国家自然科学基金资助项目(编号:30900556)

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。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 recombi- nant ING1 splicing variants plasmids were transfected into a hepatoma cell line HepG2 with lipofectamine, respec- tively. 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, p21^wafl , p14^arf, mdm2 genes were detected by quantitive reverse tran- scriptase polymerase chain reaction （RT-PCR） and Western blot. Meanwhile, the luciferase assay was performed to analyze the promoter activity of p21^wafl. In addition, Half-life time of wild type p53 protein after transfection was de- tected by western blot and protein physical binding of ING1 splicing variants with mdm2 ,p14^arf were explored by u-sing coimmunoprecipitation （IP） method. Results Overexpression of pcDNA3-1NGlb or pcDNA3-INGlc variant promoted higher hepatoma cell apotosis [ （24.36 ±0.97）%, （27.33 ± 1.12）% ,respectively] and G0/G1 arrest compared with the pcDNA3 plain vector control group [ （ 8.22± 0.86） % ] （ P 〈 0.01 ） ,while there was no signifi- cant difference between ING1 a 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, ING1 b and ING1 c significantly up-regulated the mRNA and protein expression levels of bax, p21 w^n , p l4,a, but repressed bcl-2 and mdm2 expression （P 〈 0.01 ）. INGla exerted no obvious influence on the mRNA or protein expression of bax, p21^wafl , p14^arf, bel-2 and mdm2 （P 〉 0.05 ）. The activity of p21 wan promotor was also strongly en- hanced by ING1 b or ING1 c by using luciferase reporter assay （P 〈 0.01 ）. ING1 b and ING1 c variant, but not INGla markedly prolonged the half-life of wild-type p53 and increased p53

关 键 词：生长抑制因子1 P53 凋亡 细胞周期阻滞 

分 类 号：R735.7[医药卫生—肿瘤] R730.23[医药卫生—临床医学]