Stealth核糖核苷酸干扰抑制胰腺癌细胞DNMT1表达的研究

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

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 TMRNAi 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、48 h、72 h后用荧光显微镜观察转染效率,以48 h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(Westem 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48 h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48 h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。

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.