雌激素受体GPR30 RNAi干扰载体的构建及在耐药乳腺癌细胞MCF-7R中的效果验证  

Construction of GPR30 RNAi vector and characterization in antihormone resistant breast cancer resistant cells

作　　者：赵征 刘志刚 雷光焰 党诚学[2] 廖子君 陆建荣 王云梅 苏智祥 

机构地区：[1]陕西省肿瘤医院,陕西西安710061 [2]西安交通大学医学院第一附属医院肿瘤外科,陕西西安710061

出　　处：《现代肿瘤医学》2012年第4期679-681,共3页Journal of Modern Oncology

基　　金：陕西省科技计划项目(编号:2011K13-01-14)

摘　　要：目的:采用RNA干扰(RNAi)技术研究耐药乳腺癌细胞MCF-7R中GPR30表达。方法:利用他莫昔芬(tamoxifen,TAM)诱导乳腺癌细胞MCF-7建立耐药乳腺癌细胞株。以GPR30为靶基因,pGenSil-1质粒为载体,根据GenBank数据库提供的GPR30基因核苷酸序列,克隆到空载体pGenSil-1中,Lipofectin2000转染MCF-7R细胞,G418筛选获得阳性克隆细胞扩大培养。Trizol法提取细胞总RNA,半定量RT-PCR检测GPR30的表达。结果:经TAM处理可以诱导GPR30 mRNA的表达,HindⅢ和EcoRⅠ双酶切鉴定重组干扰载体pGg30-1、pGg30-2得到了与预期大小相符的片段,RT-PCR显示,未转染组MCF-7R细胞GPR30mRNA的表达量明显高于转染pGg30-1、pGg30-2的MCF-7R细胞,而转染空载体组细胞未见明显变化。结论:RNAi技术可成功构建抑制GPR30表达的小干扰RNA重组体。Objective： To construct and identify GPR30 gene expression vector of RNA interference（RNAi） in resistant breast cancer cells.Methods： MCF-7 cells were induced to breast cancer resistant cells by Tamoxifen（TAM）,GPR30 as a target gene,pGenSil-1 plasmid as a carrier,the nucleotide acid sequences of GPR30 gene were provided by the GenBank database,which were cloned into the vector of pGenSil-1,MCF-7R cells were transfected with Lipofectin200,positive clones were screened by G418 cell expansion culture,the extraction of total cellular RNA by Trizol,and the expression of GPR30 was detected by semi-quantitative RT-PCR.Results： The treatment of TAM induced GPR30 mRNA expression,the expected size of the fragment of vector by pGg30-1,pGg30-2 was realized,which was digested by restriction enzyme of Hind Ⅲ and EcoRⅠ.RT-PCR showed that the expression of GPR30 mRNA in untransfected MCF-7R cells was significantly higher than transfected by pGg30-1 and pGg30-2 in MCF-7R cells,however,transfected cells were not changed significently in empty vector.Conclusion： The RNAi technology can construct GPR30 expression vector in breast cancer resistant cells successfully.

关 键 词：G蛋白偶联受体30 乳腺癌耐药 RNA干扰 

分 类 号：R730[医药卫生—肿瘤] R737.9[医药卫生—临床医学]