RNA干扰沉默缺氧诱导因子-1α逆转乳腺癌的耐药性  

Reversion the multidrug resistance of human breast carcinoma cells by RNA interference targeting HIF-1 alpha gene

作　　者：马超[1] 周庚寅[1] 肖颖[2] 高鹏[1] 张翠娟[1] 

机构地区：[1]山东大学医学院病理学教研室,济南250012 [2]山东大学医学院分子医学实验室,济南250012

出　　处：《中华病理学杂志》2006年第6期357-360,共4页Chinese Journal of Pathology

基　　金：国家自然科学基金资助项目(30300124)

摘　　要：目的观察RNA干扰缺氧诱导因子(HIF)-1α逆转乳腺癌耐药性的作用。方法构建靶向缺氧诱导因子-1α的短发夹状小干扰RNA基因并转染到人乳腺癌耐阿霉素细胞株MCF-7/ADR中。常规MTT法检测细胞存活率,外排实验检测细胞的P-糖蛋白转运功能,逆转录聚合酶链反应(RT-PCR)检测细胞的HIF-1α、多药耐药基因1(mdr-1)mRNA变化,Western印迹法观察干扰前后HIF-1α、P-糖蛋白的变化。结果测序证实成功构建HIF-1α的短发夹状siRNA真核表达载体pSilencer-HIF,转染空质粒ADR/neo不影响肿瘤细胞的耐药性。MTT法检测ADR/shRNA组细胞存活率由76%下降到43%,Rh123荧光强度由22·0%升为86·6%,ADR/shRNA组中HIF-1α、mdr-1mRNA、蛋白水平显著降低(P<0·05),且Spearman相关分析HIF-1α和mdr-1指数在三组中显示呈正相关(r=0·816,P<0·01)。结论成功构建了HIF-1α的短发夹状siRNA真核表达载体pSilencer-HIF,可显著降低乳腺癌MCF-7/ADR细胞中HIF-1α表达从而起到逆转肿瘤耐药的作用。Objective To reverse the multidrug resistant （MDR） phenotype of human breast carcinoma cells by small hairpin RNA （ shRNA ） technique targeting hypoxia-inducible factor（HIF） -1α gene. Methods Small hairpin RNA （shRNA） eukaryotic expression vector targeting HIF-1α gene, named pSilencer-HIF, was constructed and transfected into MCF-7/ADR human breast cancer cells by liposome technique. Tumor cell livability （TCL） and Rhodamine 123 efllux assay were used to monitor the biological changes of the transfected cells. The mRNA and protein expression of HIF-1α and mdr-1 were investigated by RT-PCR and Western blot. Results The successful construction of pSilencer-HIF plasmid was confirmed by DNA sequencing. HIF-1α mRNA and protein levels were significantly decreased in MCF-7/ADR cells after the transfection and there was a direct correlation between HIF-1α and mdr-1 expression. By comparing the cells transfected with control vector and the MCF-7/ADR cells transfected with pSilencer-HIF, a reduced TCL from 76% to 43%, and an increased Rhodamine 123 fluorescence intensity from 22.0% to 86. 6% were observed. Conclusions pSilencer-HIF-let has been successfully constructed. The inhibition of HIF-1α expression through shRNA technique can significantly reverse the multidrug resistance phenotype of MCF-7/ADR cells.

关 键 词：乳腺肿瘤 药物耐受性 糖蛋白 转录因子 低氧诱导因子-1Α 

分 类 号：R737.9[医药卫生—肿瘤]