沉默DNMT1和DNMT3b基因对胰腺癌BxPC-3细胞p16,RASSF1A基因启动子甲基化的影响

Effect of gene silencing of DNMT1 and DNMT3b on the methylation in the promoter region of p16 and RASSF1A gene in pancreatic carcinoma BxPC-3 cells

目的:探讨沉默DNMT1和DNMT3b基因对胰腺癌BxPC-3细胞p16,RASSF1A基因启动子甲基化的影响。方法:将胰腺癌BxPC-3细胞分为5组:DNMT1干扰组(转染DNMT1-siRNA),DNMT3b干扰组(转染DNMT3b-siRNA),双重干扰组(转染DNMT1+DNMT3b-siRNA),阴性对照组(转染阴性siRNA)和空白对照组(转染脂质体)。转染48 h后,应用荧光定量PCR法和Western blot法分别检测细胞中DNMT1和DNMT3b的mRNA及蛋白的表达水平;甲基化特异性PCR法检测p16和RASSF1A基因启动子甲基化。结果:与空白对照组和阴性对照组比较,各干扰组目的基因的mRNA及蛋白表达量均明显降低(均P<0.01)。空白对照组与阴性对照组p16和RASSF1A基因甲基化阳性;DNMT1干扰组和双重干扰组p16基因甲基化阴性,RASSF1A基因部分甲基化;DNMT3b干扰组p16基因部分甲基化,RASSF1A基因甲基化阳性。结论:DNMT1单干扰对胰腺癌BxPC-3细胞p16和RASSF1A基因的去甲基化作用优于DNMT3b单干扰,DNMT1和DNMT3b双重干扰无明显的协同效应;提示DNMT1是胰腺癌去甲基化治疗的一个有效靶点。

Objective： To investigate the effect of gene silencing of DNMT1 and DNMT3b on methylation in the promoter region of p16 and RASSF1A gene in pancreatic carcinoma BxPC-3 cells. Methods： The pancreatic carcinoma BxPC-3 cells were divided into five groups,which included the DNMT1 interference group（transfected with DNMT1-siRNA）,DNMT3b interference group（transfected with DNMT3b-siRNA）,double interference group（transfected with DNMT1＋DNMT3b-siRNA）,negative control group（transfected negative-siRNA） and blank control group（transfected with lipofectamine）.Forty-eight hours after transfection,the mRNA and protein expression of DNMT1 and DNMT3b were analyzed by real-time RT-PCR and Western blot,and the methylation status in the promoter region of p16 and RASSF1A gene was detected with methylation-specific PCR（MSP）. Results： Compared with the negative or blank control group,the mRNA and protein expression levels of the targeted genes were all markedly down-regulated in each interference group（all P0.01）.The methylation status of p16 and RASSF1A gene was positive in both the blank and negative control group;the p16 gene was unmethylated and RASSF1A gene was partially methylated in DNMT1 interference group and double interference group;the p16 gene was partially methylated and RASSF1A gene showed methylation-positive state in DNMT3b interference group. Conclusion： DNMT1 interference has better effect than DNMT3b interference on demethylation in BxPC-3 cells,and the double knockdown of DNMT1 and DNMT3b exerts no synergistic action.DNMT1 could serve as an effective target for the demethylation therapy of pancreatic carcinoma.