针对表皮生长因子受体基因RNA干扰质粒表达载体的构建

Construction of shRNA Expressing Plasmid Vector Inducing RNAi for EGFR Gene

目的:表皮生长因子受体(EGFR)是一种酪氨酸激酶跨膜受体,人类的多种恶性肿瘤的发生与表皮生长因子受体过度表达有关。EGFR已成为阳性表达肿瘤的重要治疗靶标。在哺乳动物细胞中,RNA干扰是通过与目的基因特异互补的21-23个硷基的小双链RNA来抑制该基因的表达。有研究表明表达小发夹RNA的载体能诱导产生更好的RNA干扰效果。本实验通过向pSIREN-Shuttle质粒插入一段可被转录成小发夹RNA的双链寡核苷酸序列(其中含有于EGFR基因序列相同的19nt序列)重构了RNAi质粒表达载体。通过细胞转染评价该载体对人鼻咽癌细胞株(HNE1)EGFR基因的mRNA表达抑制效果。结果显示HNE1细胞的EGFR基因mRNA被明显抑制。提示shRNA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： Deregulated and excessive expression of epidermal growth factor receptor （EGFR）, a transmembrane receptor tyrosine kinase, is a feature and/or cause of a wide range of human cancers, and thus inhibition of its expression is potentially therapeutic. In RNA interference （RNAi）, duplexes of 21 - 23 nt RNAs （small interfering RNA, siRNA） corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian ceils. It was approved that vectors containing shRNA expressing cassette could induce good result of RNAi. We reconstructed pSIREN - Shuttle Vector into a vector containing shRNA expressing cassette pSIREN - EGFRs to induce RNAi for EGFR gene by inserting duplex of oligonucleotides which contains 19 - nt specific sequence of EGFR. Here we showed that by using the plasmid vector pSIREN - EGFRs to induce RNAi suppressing the expression of endogenous EGFR in HNE human nasopharyngeal epidermal carcinoma ceils. As a consequence, the mRNA of EGFR was decreased, siRNA - mediated inhibition of EGFR gene induced by shRNA expressing plasmid vector may constitute a useful approach in the treatment of human nasopharyngeal epidermal cancer.