RNAi抑制DNA甲基转移酶1的表达对胃癌细胞p16基因的影响  

Effects of inhibiting the expression of DNMT1 on the p16 gene of gastric cancer cells

作　　者：赵军[1] 朱磊 赵国海[1] ZHA;ZHU Lei;ZHA(Department of Gastrointestinal Srugery, Yijishan Hospital of Wannan Medical College, Wuhu 241000, Anhui, China)

机构地区：[1]皖南医学院弋矶山医院胃肠外科

出　　处：《中国临床药理学与治疗学》2018年第4期395-399,共5页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：安徽省科技攻关计划项目(1604a0802098);芜湖市科技局科技惠关民计划项目(2014hm26)

摘　　要：目的:运用RNAi(RNA interference)抑制胃癌细胞株BGC-823中DNA甲基转移酶1(DNA methylation transferase 1,DNMT1)基因的表达,当DNMT1基因沉默后观察p16基因的表达情况。方法:以DNMT1的mRNA核苷酸序列,构建si RNA质粒1、2、3和阴性对照si RNA质粒b,胃癌细胞培养后经脂质体转染。对DNMT1基因行QPCR检测其mRNA表达水平及Western blot检测其蛋白表达水平,确认其表达下调后筛选出最佳干扰质粒。以同样的方法检测胃癌细胞最佳干扰组、阴性对照组和空白对照组的p16基因的表达情况,以确认抑制DNMT1基因表达后对胃癌细胞p16基因的影响。结果:RNAi可有效抑制胃癌细胞株BGC-823中DNMT1基因的表达。对转染后的胃癌细胞DNMT1基因检测mRNA及蛋白表达情况,结果显示,转染si RNA2的胃癌细胞中DNMT1的表达明显低于其他转染组、阴性对照组及空白对照组(P<0.05),由此筛选出最佳干扰质粒si RNA2。用同样的方法检测胃癌细胞最佳干扰组(siRNA2)、阴性对照组和空白对照组的p16基因表达情况。结果显示,最佳干扰组的mRNA(1.6727±0.2242)及蛋白(0.9227±0.0337)表达水平均高于阴性对照组(1.0025±0.0877)、(0.5440±0.0229)和空白对照组(0.4729±0.0940)、(0.4767±0.0774)(P<0.05)。结论:用RNAi可以抑制胃癌细胞DNMT1基因的表达从而使p16基因去甲基化,使p16基因恢复表达。AIM： To inhibit the expression of DNMT1 （DNA metylation transferase 1, DNMT1 ） gene in gastric cancer BGC-823 cells by RNAi and to investigate the expression of p16 gene after the silence of DNMT1. METHODS： siRNA plasmid 1,2,3 and negative control siRNA plasmid b were construtted according to the nucleotide sequence of DN-MT1. Gastric cancer BGC-823 cells were transfected with the constructed siRNA plasmid in mediation of liposome. Transcription levels of DNMT1 mRNA were determined by Q-PCR, protein expression levels by Western blot to select the best interference plasmid after the DNMT1 gene silence. Then used the same methods to investigate the expression of p16 gene in gastric cancer cells, and effects of inhibiting the expression of DNMT1 on the p16 gene of gastric cancer cells. RESULTS：The RNAi could effectively inhibit the expression of DNMT1 gene in gastric cancer BGC-823 cells. After transfection, transcription levels of DNMT1 mRNA were determined by Q-PCR, and translation levels of DNMT1 protein by Western blot. The results showed that the expression of DNMT1 in siRNA plasmid 2 group was markedly down-regulated compared to other transfection groups, negative control group and blank control group （P〈0.05）;so siRNA plasmid 2 was the best interference plasmid. Used the same methods to investigate the expression of p16 gene in the best interference group （siRNA plasmid 2）, negative control group and blank control group, the expression of p16 gene mRNA （1.6727± 0.2242 ） and protein （0.9227 ± 0.0337 ） in the best interference group was markedly up-regulated compared to the negative control group （1.0025 ± 0.0877 ）, （0.5440 ±0.0229） and blank control group （0.4729 ± 0.0940）, （0.4767 ± 0.0774）, （P 〈 0.05）. CONCLUSION： RNAi can inhibit the DNMT1 gene expression to promote the demethylation and restore expression of p16 gene.

关 键 词：胃癌 DNA甲基转移酶 RNA干扰 甲基化 

分 类 号：R965.2[医药卫生—药理学]