RNAi沉默DNMT1基因对胰腺癌细胞ppENK甲基化状态的影响

Effects of RNAi-mediated inhibitor of DNMT1 gene silencing on ppENK methylation in the pancreatic cancer cell line

目的观察RNA干扰(RNA interference,RNAi)沉默DNMT1基因的表达对胰腺癌Panc-1细胞的前脑啡肽原(preproen-kephalin,ppENK)基因甲基化状态的影响。方法采用针对DNMT1的Stealth核糖核苷酸干扰(siRNA)胰腺癌细胞,观察DN-MT1信使核糖核酸(mRNA)及DNMT1蛋白的表达;甲基化特异性PCR反应(MSP)检测不同处理组ppENK的甲基化状态。结果 Stealth RNAi转染到胰腺癌细胞Panc-1中48 h后,siRNA-3转染组的mRNA相对表达量和比率分别为0.116和32.9%,与空白对照组(0.352,100%)、脂质体对照组(0.346,98.2%)、RNAi阴性对照组(0.339,96.3%)相比差异有统计学意义(F=2.107 E4,P=0.000 1),转染组的mRNA相对抑制率为67.1%。siRNA-3转染组DNMT1蛋白相对表达量和比率分别为0.179和32.9%,与空白对照组(0.547,100%)、脂质体对照组(0.520,95%)、RNAi阴性对照组(0.535,97%)相比差异有统计学意义(F=2.195 E5,P=0.000 1)。于100 bp和96 bp处可见ppENK基因特异性条带,呈现部分去甲基化状态的特点。结论 DNMT1靶向的Stealth RNA瞬时转染到胰腺癌细胞Panc-1中可明显沉默DNMT1的基因和蛋白的表达,逆转抑癌基因ppENK的高甲基化状态,为胰腺癌的基因治疗提供相关的理论依据。

Purpose To investigate the effects of RNAi-mediated inhibitor of DNMTI gene silencing on ppENK Methylation in the pan- creatic cancer cell line Panc-1. Methods To observe the DNMT1 expression of mRNA and protein by using specific DNMT1 siRNA sequence and the ppENK methylation status was analyzed by methylation-specific polymerase chain reaction. Results The relative ex- pression quantity of DNMT1 gene mRNA and relative ratio in siRNA group were 0. 116 and 52.9% , which compared with the blank control group （0. 552, 100% ）, lipofectamine control group （0. 346, 98.2% ） and RNAi negative control group （0. 339, 96. 3% ）, the relative expression ratio of mRNA was decreased to 67.1% in the transfected group （ siRNA-3 group）. There were significant differences compared with controls （ F = 2. 107 E4, P = 0. 000 1 ）. The relative expression quantity of DNMT1 protein and relative ratio in siRNA group were 0. 179 and 32. 9% , which compared with blank control group, lipofectamine control group and negative control group were 0. 179 and 32. 9% , 0. 547 and 100% , 0. 520 and 95% , 0. 535 and 97% respectively, there were significant differences compared with control （ F =2. 195 E5, P =0. 000 1 ）. The part of restoration of ppENK gene and the demethylation can be detected in the Stealth RNAi group both at size 96 bp and 100 bp. Conclusions The RNAi targeting DNMT1 gene could down regulated the DN- MTI gene expression both on mRNA and protein level in Panc-1 cells. At the same time, demethylated the promoter of the ppENK is partly reversed. This study provides a new gene therapy approach for pancreatic cancer.