DNA甲基转移酶3b对肝癌细胞系中细胞周期素D1基因的表达和启动子甲基化的影响

目的 探讨DNA甲基转移酶3b(DNMT3b)在人肝癌细胞系(SMMC7721)中对细胞周期素D1(cylin D1)表达及其启动子甲基化水平的影响,并进一步探讨DNMT3b的作用.方法 用小分子干扰RNA(siRNA)技术抑制DNMT3b在SMMC7721细胞系中的表达(实验组),另设转染对照siRNA的对照组,及未加任何处理因素的正常组.用蛋白质印迹检测转染前后DNMT3b及cylin D1蛋白的表达.用噻唑盐(MTT)法检测其生长增殖的变化,并应用甲基化特异性PCR(MSP)技术分别检测实验、对照两组细胞中cylin D1基因启动子区的甲基化状况.结果 DNMT3b siRNA 转染组SMMC7721的生存率(53.7%)与对照组、正常组相比明显降低(P=0.01).蛋白质印迹结果 显示DNMT3b表达水平明显低于对照组和正常组,cylin D1蛋白的表达也低于对照组和正常组.两组中cylin D1启动子区甲基化状态无差异,均未发生甲基化.结论 DNMT3b可以调节cyclinD1的表达,而不改变基因的甲基化状态,可能发挥了转录调控因子的作用.

Influence of DNA methyltransferase 3b on the expression of cylin D1 gene and methylation of its promoters in human hepatocellular carcinoma cells

Objeetive To investigate the influence of DNA methyltransferase(DNMT)3b on the expression of cylin D1 gene and methylation of its promoters and to investigate the function of DNMT3b.Methods Human hepatocellular carcinoma cells of the line SMMC7721 were cuhured and randomly divided into 3 groups:experimental group transfected with siRNA to silence the DNMT3b,control group transfected with control siRNA,and normal group without transfection.The transfection rate of siRNA was detected by fluorescence microscopy.MTr method was used to measure the survival rate of the SMMC-7721 cells.Western blotting and cell proliferation assay were performed to evaluate the expression of cyclin D1 and cell growth.Methylation specific PCR(MSP)was performed to investigate whether the promoter of cyclin D1 was methylated.Results F1uorescence microscopy showed that the transfection rate of siRNA was over 90%.MTr method showed that 24 h and 36 h after transfection the A value and survival rate of the SMMC7721cells of the experimental group were both significantly higher than those of the control d normal groups(all P＜0.05).Western blotting showed that the expression levels of DNMl3b and cyclin D1 of the experimental groud decreased significantly compared with the control and the normal groups.MSP showed no obvious change of the state of methylation among the 3 groups.Condusions DNMT3b may regulate the expression and the function of cyclin D1 gene in the human hepatocellular carcinoma cells,but does not change its methylation state.DNMT3b may play their role as a signal transduction element rather than as a DNA methyltransferase.