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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 effects of DNMT1 and DNMT3b silencing on promoter methylation of p16 and RASSF1A genes in pancreatic cancer BxPC-3 cells. Methods: The pancreatic cancer BxPC-3 cells were divided into 5 groups: DNMT1 interference group (transfected with DNMT1-siRNA), DNMT3b interference group (transfected with DNMT3b-siRNA), double interference group (transfected with DNMT1 + DNMT3b-siRNA) Control group (transfected with negative siRNA) and blank control group (transfected with liposomes). 48 h after transfection, the mRNA and protein expression levels of DNMT1 and DNMT3b were detected by fluorescence quantitative PCR and Western blot respectively. The methylation of p16 and RASSF1A promoter was detected by methylation-specific PCR. Results: Compared with the blank control group and the negative control group, mRNA and protein expression of the target gene in each interference group were significantly decreased (all P <0.01). The methylation of p16 and RASSF1A genes in the blank control group and the negative control group was positive. The methylation of p16 gene in DNMT1 interference group and double interference group was negative, and RASSF1A gene was partially methylated. The DNMT3b interference group partially methylated p16 gene and RASSF1A gene A Base-positive. CONCLUSION: DNMT1 single-knockdown is more effective than DNMT3b in demethylation of p16 and RASSF1A genes in pancreatic cancer BxPC-3 cells. There is no obvious synergistic effect between DNMT1 and DNMT3b double knockdown. It suggests that DNMT1 is a demethylating agent for pancreatic cancer A valid target.