抑制表皮生长因子受体基因表达的pSIREN-Shuttle RNAi表达载体的构建  

Construction of RNAi expressing plasmid vector of pSIREN-shuttle for EGFR gene silencing

作　　者：栾建刚[1] 梁传余[2] 文艳君[3] 李炯[3] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院耳鼻咽喉科,山东烟台264000 [2]四川大学华西医院耳鼻咽喉科,四川成都610041 [3]四川大学华西医院国家重点生物治疗实验室,四川成都610041

出　　处：《山东大学耳鼻喉眼学报》2006年第1期4-8,共5页Journal of Otolaryngology and Ophthalmology of Shandong University

摘　　要：目的:构建抑制表皮生长因子受体(EGFR)基因表达的RNA干扰(RNAi)质粒表达载体pSI-REN-Shuttle-EGFR。方法:化学合成3条编码短发夹RNA序列的、特异靶向EGFR基因的寡核苷酸链,各69个碱基,退火,克隆到RNAi-Ready pSIREN-Shuttle载体的人源U6启动子的下游,重组构建RNAi质粒,3种载体转染人鼻咽癌细胞株(CNE),用RT-PCR分析pSIREN-Shuttle-EGFR载体对EGFR基因的mRNA表达抑制效果。结果:重组构建的pSIREN-Shuttle-EGFR载体经插入基因片段序列分析,表明69个碱基成功插入到预计位点,并且序列完全一致。CNE细胞EGFR基因的mRNA被明显抑制。结论:RNAi质粒表达载体介导对EGFR基因的RNA干扰可能是鼻咽癌干预治疗的一种有效手段。Objective： To construct an expressing vector （pSIREN-Shuttle-EGFR） of RNAi in order to suppress gene expression of epidermal growth factor receptor （EGFR）. Methods： Three 69 base-pair oligos coding hairpin RNA which specifically aimed at EGFR gene were chemically synthesized and annealed, then inserted into the downstream of U6 promoter of vector to construct RNAi plasmid （pSIREN-Shuttle-EGFRs）. Sequence analysis was used to identify the correction of recombinant vector. RT-PCR was performed on the CNE cell line transfected by pSIREN-Shuttle-EGFRs. Results： Sequencing analysis demonstrated that 69 bp had been inserted into expected site and the insertion sequence was exactly correct in recombinant pSIREN-Shuttle-EGFRs vectors. It was showed that using the plasmid vector pSIREN-EGFRs to induce RNAi suppressed the expression of endogenous EGFR in CNE human nasopharyngeal epidermal carcinoma cells. As a consequence, the mRNA of EGFR was decreased. Conclusion： RNAi-mediated inhibition of EGFR gene induced by shRNA expressing plasmid vector may constitute a useful approach in the treatment of human nasopharyngeal epidermal cancer.

关 键 词：受体 表皮生长因子 RNA 信使 RNA 干扰 RNA 小发夹 

分 类 号：Q782[生物学—分子生物学] R739.63[医药卫生—肿瘤]