针对MTA1基因siRNA载体构建和沉默效应  

Construction of a siRNA vector targeting human MTA1 gene and the gene-silencing effect

作　　者：杨松华[1] 赵国强[1] 董子明[1] 

机构地区：[1]郑州大学基础医学院,河南郑州450001

出　　处：《南方医科大学学报》2008年第3期366-369,共4页Journal of Southern Medical University

基　　金：教育部"十五"211工程重点学科建设项目(教重办2002第2号)

摘　　要：目的构建针对人MTA1基因的小干扰RNA(siRNA)表达载体,观察RNA干扰对食管癌细胞MTA1基因表达的抑制作用。方法设计MTA1的siRNA靶序列,分别合成两条互补的寡核苷酸链,退火后重组入pRNAT-U6.2载体,转化扩增后进行序列测定。用脂质体包裹转染人食管癌细胞EC9706,采用RT-PCR和Westernblotting分别检测MTA1基因mRNA和蛋白表达的变化。结果把针对MTA1基因的siRNA的双链寡核苷酸片段克隆入pRNAT-U6.2载体,经测序分析,插入片段正确;RT-PCR和Westernblotting检测显示,MTA1基因的表达水平明显降低,其中以481-499(GACCCTGCTGGCAGATAAA)为靶序列的siRNA沉默作用最强,MTA1蛋白几乎完全不表达。结论成功构建出沉默食管癌细胞MTA1基因表达的siRNA载体,为通过沉默MTA1基因的表达来减少恶性肿瘤侵袭转移提供了实验基础。Objeetlve To construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells. Methods The siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively. Results The double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA （481-499）, which induced almost complete silencing of MTA1 protein expression. Conclusion The siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.

关 键 词：RNA干扰 MTA1 食管癌 

分 类 号：R73-3[医药卫生—肿瘤]