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论著: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
卜淑蕊 钱家鸣
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作者单位:中国医学科学院北京协和医学院
中文关键字:胰腺癌;RNA干扰;甲基化转移酶1
英文关键字:Pancreatic cancer; RNA interfering(RNAi); DNA methyltransferase1 (DNMT1)
中文摘要: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、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(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。
英文摘要: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 3rd 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.
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