U6/H1双启动子siRNA表达框架构建及其对肿瘤细胞端粒酶活性的干扰作用  

Construction of U6/H1 dual promoter siRNA expression cassettes and their interfering effects on telomerase activity in tumor cells

作　　者：龚霞[1] 张慧慧[1] 彭剑雄[1] 罗建新[1] 

机构地区：[1]中南大学湘雅医学院医学检验系,湖南长沙410013

出　　处：《中国普通外科杂志》2014年第6期769-774,共6页China Journal of General Surgery

基　　金：湖南省科技厅课题资助项目(98SSY1003)

摘　　要：目的:构建针对端粒酶hTERT基因的U6/H1双启动子siRNA表达框架(SEC),并观察其转录产物对HeLa细胞端粒酶活性的干扰作用。方法:利用融合PCR技术,针对人端粒酶hTERT基因开放阅读区构建3条U6/H1双启动子SEC以及针对其3'端非翻译区构建1条U6/H1双启动子SEC,对各SEC鉴定后,分别转染人宫颈癌HeLa细胞,用端粒重复序列扩增法(TRAP)检测HeLa细胞的端粒酶活性。结果:4种针对端粒酶hTERT基因的U6/H1双启动子SEC均成功构建,转染HeLa细胞后,对端粒酶活性的抑制率分别为36.8%、57.39%、80.47%、70.31%。结论:针对端粒酶hTERT基因的U6/H1双启动子SEC的成功构建,为开展肿瘤端粒酶基因干扰的实验研究提供了新的有效手段。Objective： To construct the U6/H1 dual promoter siRNA expression cassettes （SEC） targeting human telomerase hTERT gene, and to observe the interfering effect of their transcription products on hTERT geneactivity in HeLa cells. Methods： By fusion PCR, three U6/H1 dual promoter SECs targeting the open reading frame of the hTERT geneand one SEC targeting the 3＇ untranslated region of the hTERT gene were constructed. The SECs were transferred into human cervical cancer HeLa cells respectively after identification, and then the telomerase activity in theHeLa cells was assessed by telomeric repeat amplification protocol （TRAP）. Results： The four SECs targeting human telomerase hTERT gene were all successfully constructed, and aftertheir transfection into the HeLa cells, the rate of telomerase activity inhibition was 36.8%, 57.39%, 80.47%, and70.3196, respectively. Conclusion： The successful construction of SECs targeting human telomerase hTERT gene may provide a noveleffective approach for study of the gene interference of tumor telomerase.

关 键 词：端粒 末端转移酶 RNA干扰 启动区(遗传学) 

分 类 号：R730.5[医药卫生—肿瘤]