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

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。

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

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 usingthe 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.