DNMT1、DNMT3b siRNA重组质粒转染对人膀胱癌细胞DNMTs表达的影响及其生物学效应  

The Eexpression Levels of DNMTs and Biological Effects after Down-regulating DNMT1 and DNMT3b Gene Expression in Human Bladder Carcinoma Cells

作　　者：吴振启[1,2] 张旭[3] 刘毅[1] 徐华[4] 史涛平[1] 权伟合[2] 李虎宜[2] 张有顺[5] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030 [2]郧阳医学院附属东风总医院泌尿外科 [3]北京301医院泌尿外科 [4]武汉大学分子生物学实验室 [5]郧阳医学院附属东风总医院分子生物学实验室

出　　处：《临床泌尿外科杂志》2010年第2期142-146,共5页Journal of Clinical Urology

摘　　要：目的:探讨DNMT1、DNMT3b基因在膀胱癌发生发展中的作用。方法:①将DNMT1、DNMT3bsiRNA重组质粒分别单独和共同转入人膀胱癌细胞系BIU-87;②半定量RT-PCR和Western Blot分别检测DNMT1和DNMT3b基因mRNA和蛋白水平的变化;③用MTT法观察各组细胞生长曲线,流式细胞术观察细胞生长周期及凋亡率的变化。结果:单独和共同转染DNMT1(或DNMT3b)mRNA、蛋白表达量明显下降,能影响BIU-87的生长曲线,使细胞增殖减慢,并使细胞周期阻滞于G_1期,明显提高细胞调亡率;转染空质粒和单独转染DNMT3b siRNA重组质粒对BIU-87细胞的生长和增殖无明显影响。结论:联合转染DNMT1、DNMT3b siRNA重组质粒,可同时下调DNMT1和DNMT3b在BIU-87细胞中的表达,并能在体外抑制BIU-87细胞生长,促进细胞凋亡发生,其效果优于单独转染的重组质粒。单独转染DNMT3b siRNA重组质粒对BIU-87细胞的生长和增殖无明显影响。Objective：To study the effects of DNMT1 and DNMT3b gene in occurrence and development of bladder carcinoma. Methods：（1） The constructed DNMT1,DNMT3b siRNA recombinant plasmids were single or both transfected into BIU-87 cells. （2）The expression levels of DNMT1 and DNMT3b protein and mRNA were detected by Western Blot and RTPCR analysis after transfection. （3）The proliferation ability of different transfection cell groups was observed by MTT method. Flow cytometry was used to detect the change of the cell cycle and the apoptosis rate. Results：（1）The result demonstrated after transfection of the DNMT1 and/or DNMT3b gene siRNA recombinant plasmids. （2）Both co-transfection group and single transfection of antisense DNMT1 （or DNMT3b） gene iRNA recombinant plasmids decreased the protein（or mRNA） level（s） of DNMT1 （or DNMT3b） significantly, affected the cell growth curve and blocked the cell cycle at G1 phase and increased the apoptosis rate respectively, Transfection of the DNMT3b gene siRNA recombinant plasmid or Neo-shNC recombinant plasmid did not suppress the growth and proliferation of BlU-87. Conclusions： Co-transfection of DNMT1 gene and DNMT3b gene siRNA recombinant plasmid can down-regulate the expression levels of DN MT1 and DNMT3b, and can suppress the growth of human bladder carcinoma cell line BIU-87 and induce apoptosis. It has more effect on the cell growth than single transfection of the DNMT1 gene siRNA recombinant plasmid. Transfeetion of the DNMT3b gene siRNA recombinant plasmid or Nec-shNC recombinant plasmid did not suppress the growth and proliferation of BIU 87.

关 键 词：膀胱癌 增值 凋亡 甲基化 

分 类 号：R737.14[医药卫生—肿瘤]