HER4基因siRNA载体的构建及其沉默作用  

Vector construction and silencing effect of HER4 gene targeted small interfering RNA

作　　者：李新宇[1] 逄旭光[2] 葛棣[2] 宁艳霞[3] 曾亮[2] 

机构地区：[1]福建医科大学附属泉州第一医院肿瘤外科,泉州362000 [2]复旦大学附属中山医院胸外科,上海200032 [3]复旦大学上海医学院生理与病理生理学系,上海200032

出　　处：《医学研究生学报》2010年第1期30-34,共5页Journal of Medical Postgraduates

基　　金：福建省科技项目计划(2008J0311)

摘　　要：目的RNA干扰是由双链RNA引发的转录后基因沉默。为了研究表皮生长因子受体对食管癌细胞的作用,文中构建针对人HER4基因的小干扰RNA(siRNA)及其表达载体,转染人食管癌细胞Eca-109,观察其基因沉默效果,探索食管癌基因治疗的新途径。方法根据siRNA设计原则,设计针对HER4基因的两条寡核苷酸链,退火后与载体SUPER.neo+gfp连接,然后进行酶切鉴定和DNA序列测定。用脂质体包裹转染食管癌细胞Eca-109,用荧光定量PCR和Western blot方法检测HER4蛋白表达情况。结果经酶切鉴定和DNA测序分析后,提示HER4靶向RNA干扰重组载体构建成功。转染食管癌细胞后,用荧光定量PCR和Western blot法检测HER4基因表达水平显著降低。结论成功地构建了针对人HER4基因的siRNA表达载体,并可有效抑制食管癌细胞Eca-109中该基因的表达。Objective RNA interference refers to post-transcriptional gene silencing caused by double strands RNA. To investigate the effect of EGFR receptor on esophageal carcinoma, the expression vector of HER4 gene targeted small interfering RNA was constructed to observe its silencing effect in human esophageal carcinoma cell line Eca - 109, in order to find a promising method for the gene therapy of this disease. Methods Two complementary oligo DNA strands targeting HER4 gene were designed and synthesized according to the principles of designing siRNA. After annealing, oligo DNAs were inserted into SUPER. neo ＋ gfp vector, then enzyme digestion analysis and DNA sequencing were applied. After transfecting it into human esophageal carcinoma cell line, we detec- ted the level of expression of HER4 gene through real-time quantitative PCR and Western Blot. Results The enzyme digestion analysis and DNA sequencing show that HER4 gene targeted small interfering RNA and its expression vector were constructed successfully, and after transfection, the expression of HER4 gene in esophageal carcinoma cell line was suppressed greatly. Conclusion HER4 gene targeted small interfering RNA and its expression vector were constructed successfully, and could decrease the expression of HER4 gene in Eca-109 cell line, which laid the foundation for the following experiment.

关 键 词：HER4 RNA干扰 食管癌 细胞 

分 类 号：Q782[生物学—分子生物学]