联合基因Survivin、CDK1的shRNAs靶向干扰对鼻咽癌CNE-2细胞增值的影响  

Influence of Tandem Gene Survivin and CDK1 shRNAs Expression Vector on Nasopharyngeal Carcinoma Cell Line CNE-2 Growth

作　　者：陈淑萍[1] 周红桃[2] 蔡俊宏[3] 王福利[3] 许茂轩[1] 符生苗[3] 

机构地区：[1]海南大学农学院,海口570228 [2]南方医科大学珠江医院医学检验中心,广州510280 [3]海南省人民医院医学检验中心,海口570311

出　　处：《基因组学与应用生物学》2014年第2期239-247,共9页Genomics and Applied Biology

基　　金：国家自然科学基金项目(81060223)资助

摘　　要：为了构建具有干扰效应的靶向Survivin和CDK1基因及两者串联的短发夹样RNA(short hairpin RNA,shRNA)表达载体,并研究其靶向干扰对鼻咽癌细胞CNE-2生物学行为的改变。本研究通过构建pU6-SurvivinshRNA、pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重组载体,用脂质体Lipofectamine 2000法将其转染入CNE-2细胞株中,应用RT-PCR方法初步筛选具有干扰效应的重组载体,逆转录实时荧光定量PCR(RT-qPCR)检测Survivin、CDK1 mRNA的基因表达水平,Western blot检测Survivin和CDK1蛋白表达以及噻唑蓝溴化四唑(methyl thiazolyl tetrazolium,MTT)检测癌细胞增殖活性的变化。结果表明:成功构建具有干扰效应的shRNAs表达载体;联合或单基因Survivin、CDK1的shRNAs表达载体干扰CNE-2细胞后,其对应的mRNA和目的蛋白表达均降低,CNE-2癌细胞增殖活性降低,联合基因shRNAs表达载体具有一定程度的干扰增强作用。This study was to construct the vectors to lock down the expression of Survivin and/or CDK1 by RNA interference and study the effect to the biological behavior of nasopharyngeal carcinoma cell line CNE-2. The pU6-SurvivinshRNA, pU6-CDK1shRNAand pU6-SurvivinshRNA-CDK1shRNAwere constructed and transfected to the CNE-2 cell line by Lipofectamine 2000. The changes of the expression of Survivin and/or CDK1 caused by transfecting with those vectors were detected by RT-qPCR and Western Blot. The influence to cell proliferation was determined by methyl thiazolyl tetrazolium（MTT）. Those results show that the interference effects of shRNAs expression vectors were successfully constructed and the expression levels of Survivin and/or CDK1 were declined by transfecting with them. The growth rate of CNE-2 cell line caused by transfecting with those vectors was decreased. The tandem gene Survivin and CDK1 shRNAs expression vector can more effectively inhibit the expression of Survivin and/or CDK1 than single gene Survivin or CDK1 shRNA vector.

关 键 词：短发夹样RNA Survivin基因 CDK1基因 鼻咽癌 CNE-2 

分 类 号：R739.63[医药卫生—肿瘤]