稳定低表达DNA甲基转移酶3b基因对膀胱癌细胞生长和凋亡的影响  

Effect of stable DNA methyltransferase 3bknockdown on proliferation and apoptosis in bladder cancer cells in vitro

作　　者：陈柯[1] 李炳坤[1] 许凯[1] 徐啊白[1] 刘春晓[1] 郑少波[1] 徐亚文[1] 贾晨尧 刘奇[1] 李虎林[1] 

机构地区：[1]南方医科大学珠江医院泌尿外科,广东广州510282

出　　处：《南方医科大学学报》2015年第11期1524-1529,共6页Journal of Southern Medical University

基　　金：广东省自然科学基金(S2012010009383;S2013040016823)

摘　　要：目的探讨稳定低表达DNA甲基转移酶3b(DNMT3b)基因对人膀胱癌细胞生长和凋亡的影响。方法包装表达DNMT3b si RNA和阴性对照si RNA的慢病毒,通过慢病毒感染的方法获得稳定低表达DNMT3b的膀胱癌细胞BIU-87及对照细胞。MTT和流式细胞仪检测DNMT3b稳定低表达对细胞增殖和凋亡的影响;体内裸鼠成瘤实验观察DNMT3b稳定低表达对肿瘤的抑制效果;荧光定量PCR和Western blot检测DNMT3b稳定低表达对细胞生长和凋亡相关基因表达的影响;甲基化特异性PCR检测对细胞生长和凋亡相关基因甲基化的影响。结果荧光定量PCR和Western blot检测结果显示,DNMT3b m RNA和蛋白水平在BIU-87稳定细胞株中稳定低表达;DNMT3b稳定低表达可以抑制BIU-87细胞的生长,并且抑制裸鼠皮下瘤体的生长;DNMT3b稳定低表达可以促进BIU-87细胞的凋亡;DNMT3b稳定低表达可以增加凋亡及细胞生长相关基因DAPK,Bax和RASSF1A m RNA和蛋白水平的表达;DNMT3b稳定低表达可以减少凋亡及细胞生长相关基因DAPK,Bax和RASSF1A启动子区域的甲基化水平。结论 DNMT3b稳定低表达可能通过调控凋亡相关基因以及细胞生长相关基因启动子区域的甲基化水平来影响基因的表达,从而抑制BIU-87细胞的生长,诱导细胞凋亡。Objective To investigate the effect of stable knockdown of DNA methyltransferase 3b（DNMT3b） on the proliferation and apoptosis of bladder cancer cells. Methods Lentivirus expressing DNMT3 b si RNA or the negative control si RNA was infected in human bladder cancer BIU-87 cells. MTT assay and flow cytometry were used to detect cell proliferation and apoptosis, respectively. The inhibitory effect of DNMT3 b knockdown on xenograft tumors in nude mice was observed.Real-time PCR and Western blotting were carried out to investigate the expression level of cell apoptosis related genes.Methylation specific PCR was used to examine the methylation in the promoter region of the cell apoptosis related genes.Results The results of real-time PCR and Western blotting showed that DNMT3 b m RNA and protein level were stably knocked down in BIU-87 cells. Stable DNMT3 b knockdown suppressed BIU-87 cell growth and the tumor formation ability of the cells in nude mice. DNMT3 b knockdown promoted the apoptosis of BIU-87 cells, increased the m RNA and protein expression of the cell growth and apoptosis related genes including DAPK, Bax and RASSF1 A, and significantly decreased the methylation of these genes. Conclusion Stable DNMT3 b knockdown can affect the methylation of the cell growth and apoptosis related genes to regulate their expression, which might be a possible mechanism for suppressed cell growth and enhanced apoptosis of BIU-87 cells.

关 键 词：膀胱癌 DNMT3B 稳定细胞株 甲基化 

分 类 号：R737.14[医药卫生—肿瘤]