靶向人MGMT的shRNA干扰载体的构建与鉴定  

Construction and identification of short hairpin RNA interference vector targeting human MGMT gene

作　　者：胡颖熹 冯城婷 陶敏[1] 梁容瑞[1] 赵晶 李玮[1] 史悦[1] 石凤灵 李大鹏[1] HU Yingxi;FENG Chengting;TAO Min;LIANG Rongrui;ZHAO Jing;LI Wei;SHI Yue;SHI Fengling;LI Dapeng(Department of Oncology,the First Affiliated Hospital of Soochow University,Suzhou,215000)

机构地区：[1]苏州大学附属第一医院肿瘤科,江苏苏州215000

出　　处：《现代仪器与医疗》2018年第6期50-53,共4页Modern Instruments & Medical Treatment

基　　金：国家自然科学基金;编号:81402477

摘　　要：目的:有效构建针对人MGMT(hMGMT)基因的short hairpin RNA(shRNA)质粒表达载体,转染肝癌SMMC-7721细胞,验证shRNA对肝癌SMMC-7721细胞MGMT基因的干扰效果,进一步研究hMGMT的功能及临床意义。方法:设计针对hMGMT mRNA靶序列的shRNA,PCR扩增获得目的产物,通过TA克隆目的产物与酶切后的载体片段进行连接,连接后的重组载体转化到大肠杆菌,提取质粒后送测序鉴定。脂质体包裹鉴定后的重组载体,转染肝癌SMMC-7721细胞,利用Real-Time PCR及Western Blot检测RNA干扰效果。结果:重组克隆经菌落PCR及测序证实插入的序列正确, RNA干扰载体转染肝癌SMMC-7721细胞后细胞内MGMT的mRNA及蛋白水平均显著降低。结论:所制备的hMGMT-shRNA在肝癌SMMC-7721细胞中可获得高效转染,并能产生特异性的基因沉默效应,针对hMGMT为靶标的shRNA载体能够有效下调肝癌细胞中MGMT基因的表达,为研究MGMT蛋白的功能以及临床肿瘤合理用药提供思路。Objective:The aim of this study was to effectively transfect human MGMT(hMGMT)short hairpin RNA plasmid into hepatoma cells and verify the function of MGMT gene in SMMC-7721 cells.Methods:Two small hairpin RNAs targeting hMGMT mRNA were designed and synthesized,then linked to the expression vector pENTR.The recombinant plasmid was identified by DNA sequencing and encapsulated in liposomes.Transfect the recombinant plasmid into SMMC-7721 hepatoma cells.The interference effects of MGMT were assayed by Real-Time PCR and Western Blot.Results:The recombinant clone was successfully verified by PCR amplification and sequencing.The expression of MGMT gene in SMMC-7721 cells was significantly decreased after the transfection of RNA interference vector.Conclusion:The hMGMT-shRNA plasmid expression vector targeting MGMT can inhibit the expression of MGMT at mRNA and protein levels.This study establishes a foundation for researching the function of DNA repair protein MGMT,and provides ideas for the rational uses of anticancer drugs.

关 键 词：SHRNA hMGMT 基因沉默 肝癌细胞 

分 类 号：R733[医药卫生—肿瘤]