HIF-1α基因RNA干扰质粒在胃癌细胞中的构建与鉴定

Construction of a vector carrying a small hairpin RNA targeting the HIF-1α gene and its expression in gastric cancer cells

目的:构建转录因子HIF-1α表达的RNA干扰(RNA interference,RNAi)质粒,为研究HIF-1α参与的细胞信号通路及以HIF-1α为靶点的基因治疗提供稳定转染细胞的RNAi.方法:选取HIF-1α基因的19nt特异性序列,设计针对HIF-1α的shRNA序列,应用基因重组技术克隆到pSilencer1.0-U6表达载体中,利用EcoRⅠ和ApaⅠ双酶切和DNA测序鉴定重组克隆.低氧条件下,将包含HIF-1α基因RNAi序列的重组载体转染人胃癌细胞SGC-7901,转染48h后,收集细胞裂解液,Western blotting分析低氧条件下对照组与转染组HIF-1α蛋白的表达水平.结果:双酶切证实shRNA插入pSilencer1.0-U6质粒,DNA测序证实插入的序列正确;HIF-1α基因RNAi质粒转染胃癌细胞SGC-7901成功;Western blotting结果显示与空质粒对照组比转染组细胞HIF-1α表达下调.结论:成功构建人HIF-1α基因RNAi质粒,为研究HIF-1α参与的细胞信号通路提供了稳定转染细胞的干扰质粒.同时,阻断HIF-1α的信号通路将为肿瘤基因治疗提供一个较好的靶点,研究结果为以HIF-1α为靶向的基因治疗提供了有利工具.

AIM： To construct a plasmid carrying a small hairpin RNA （shRNA） targeting the HIF1α gene and to observe its expression in gastric cancer SGC-7901 cells. METHODS： SHIF-1α-specific shRNA sequence was designed, synthesized and cloned into the expression vector pSilencer 1.0-U6. The recombi- nants were identified by double digestion with EcoR I ＋ Apa I and DNA sequencing, and thentransfected into SGC-7901 cells under hypoxia. Forty-eight hours after transfection, HIF1α expression was detected by Western blotting. RESULTS： Restriction enzyme digestion and DNA sequencing confirmed that the shRNA was correctly inserted into the plasmid pSilenc- er 1.0-U6. The recombinant plasmid carrying the shRNA targeting HIF1α was successfully transfected into SGC-7901 cells. Western blot analysis suggested that HIF1α expression was knocked down in transfected cells compared to control cells. CONCLUSION： A plasmid carrying a shRNA tar- geting HIF1α has been constructed successfully, and SGC-7901 cells stably transfected with this re- combinant vector have been obtained. These will offer favorable tools for studying HIF1α-targeted gene therapy.