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 plasraid 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 Iipofectin200, 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 M and EcoR I . 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.%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,根批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.Tnzol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.
展开▼
