Stealth核糖核苷酸干扰抑制胰腺癌细胞DNMT1表达的研究

目的:研究靶向甲基化转移酶1(DNMT1)基因的Stealth核糖核苷酸干扰(RNAi)技术对胰腺癌细胞Panc-1中DNMT1表达的抑制作用.方法:通过Block-IT TM RNAi Designer在线设计合成针对DNMT1的Stealth核糖核苷酸(siRNA),以50 nM siRNA用脂质体转染法转染Panc-1细胞.同时设空白对照组,RNAi阴性对照组,荧光RNAi组,每组3个复孔；在转染24 h、48h、72h后用荧光显微镜观察转染效率,以48h荧光强度最亮.应用逆转录聚合酶链反应(RT- PCR)检测DMNT1信使核糖核酸(mRNA)含量,免疫印迹(Western blot)检测DNMT1蛋白的表达.结果:将靶向DNMT1基因Stealth RNAi转染到胰腺癌细胞Panc-1中后,Panc-1细胞在转染RNAi后48h的转染效率为50％～60％；DNMT1的蛋白和mRNA水平显著下调,与对照组相比,转染后48h的相对抑制率分别为68％和67.8％(P＜0.05),也显著低于空白对照组及空载体组(P＜0.05).结论:DNMT1靶向的Stealth RNA瞬时转染到胰腺癌细胞Panc-1中可显著沉默DNMT1的基因和蛋白的表达,为胰腺癌的基因治疗提供新的理论依据.

Study of DNMT1 Stealth RNA Interference on the Expression of DNMTI Gene in the Pancreatic Cancer Cell Line

Objective：To investigate the suppression effect of the stealth RNAi targeting DNMT1 gene by transecting the pancreatic cancer cell line.Methods：The DNMT1-Stealth RNA was designed on line and synthesized by the Invitrogen Company.The Panc-1 cell line was transfected by the stealth RNAi in 50nM concentration of the lipofectamine 2000.The fluorescence of each group was observed under the microscope after 24 h,48 h and 72 h respectively.The expression of DNMT1 mRNA was detected by RT-PCR;the expression of DNMT1 protein was detected by Western blotting after transfected Stealth RNAi for 48 hours.Results：Forty-eight hours after the Panc-1 cell transfected by stealth RNAi,the efficiency of the transfection was between 50%to 60%.The expression of mRNA and protein of the 3~（rd） RNAi transfected group decreased 67.8%and 68%respectively comparing with the control（P 0.05）.Conclusions： The RNAi targeting DNMT1 gene is transfected to the Panc-1 cell line,which could down regulated the DNMT1 gene expression both on mRNA and protein.This study provides a new gene therapy approach for pancreatic cancer management in the future.