STAT5A基因表达对人肝癌HepG2细胞增殖和凋亡影响的观察  

Effects of cotransfection of two sites of STAT5A-targeting shRNA expression vectors on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2

作　　者：曾永秋[1] 杨华秀[2] 曹洋[3] 刘岚[1] 税青林[1] 

机构地区：[1]泸州医学院医学细胞生物学与遗传学教研室,四川泸州646000 [2]泸州医学院附属中医院肝病科,四川泸州646000 [3]泸州医学院生理学教研室,四川泸州646000

出　　处：《中华肿瘤防治杂志》2013年第1期31-35,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：四川省教育厅资助(07ZC028)

摘　　要：目的:研究抑制STAT5A基因的表达对人肝癌HepG2细胞增殖和凋亡的影响,为其作为肝癌基因治疗的新分子靶点提供实验依据,并对RNAi实验方法的优化进行探讨。方法:构建2个针对STAT5A基因不同位点的RNA干扰(RNAi)真核表达载体,为减少脱靶效应和细胞毒性,采用两载体共转染的方法转染人肝癌细胞HepG2,分别采用半定量RT-PCR、蛋白质印迹法检测转染前后细胞中STAT5A mRNA和蛋白表达的变化;采用MTT法检测细胞增殖情况;流式细胞仪检测细胞凋亡率。结果:酶切及测序结果显示,STAT5A基因两位点的shRNA表达载体均构建成功;两载体共转染HepG2细胞48h后,与对照组相比,RT-PCR结果显示实验组细胞中STAT5A mRNA的抑制率约为66%,P<0.05;蛋白质印迹法检测结果显示,STAT5A蛋白的抑制率约为58%,P<0.05;同时MTT法显示实验组较对照组在转染后24～96h内细胞生长速度减慢、增殖抑制明显(P<0.05),流式细胞仪检测实验组细胞凋亡率为(34.68±0.78)%,明显高于阴性对照组(5.12±1.09)%和空白组(4.75±1.65)%,P<0.05。结论:成功构建了针对STAT5A基因的RNA干扰真核表达载体,两载体共转染可有效抑制STAT5A基因的表达,并能抑制细胞生长、诱导细胞凋亡。OBJFA2WIVE：To study the effect of suppression of the expression of STAT 5A gene on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2, evaluate the feasibility for STAT 5A as a molecular target of gene therapy for hepatocellular carcinoma,and explore the optimization of RNAi experiment method. METHODS： Two sites of RNA interference eukaryotic expression vectors specific for STAT 5A gene were constructed and co-transfected into the HepG2 cells so as to reduce the off-target effect and cytotoxic. The expression of STAT 5A mRNA and its protein were detected by semi-quantitative reverse transcript polymerase chain reaction （RT-PCR） and immunoblotting assay （Western blot）. MTT method was used to detect the cell proliferation, and FCM was used to detect the cell apoptosis. RESULTS：Two sites of RNA interference eukaryotic expression vectors specific for STAT 5A gene were confirmed to be successfully constructed by reconstriction enzymes and DNA sequencing. Contrasted with control group,the inhibitory ratios of STAT 5A mRNA and protein in the experimental group were about 66O/oo and 58% respectively at 48 h after co-ntransfection（P〈0.05）. The result of analysis of MTT showed that the cell growth velocity of the experiment group was step down and the proliferation inhibition rate was inhibited significantly after 24-96 h of transfection（P〈0. 05 ）. FCM assay indicated that the apoptotic rate of the experiment group was （34.68±0.78） %, which was signficantly higher than that of negative control group （5.12 ±1.09 ）% and blank group （4.75 ± 1.65 ）% （P〈 0.05 ）. CONCLUSION： RNA interference eukaryotic expression vectors targeted at STAT 5A were successfully constructed, and cotransfection of the two vectors can inhibit the expression of STAT 5A gene efficiently,inhibit the proliferation of HepG2 celhand promote apoptosis obviously.

关 键 词：肝肿瘤 STAT5A基因 RNA干扰 HEPG2细胞 细胞增殖 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]