miRNA-7真核表达载体的构建与鉴定

Construction and identification of eukaryotic expression vector encoding miRNA-7

目的构建miR-7真核表达载体并观察其对人肺癌细胞体外生长的影响。方法以人肺癌细胞系95D细胞基因组DNA为模板,PCR法扩增miR-7前体序列,经BamHⅠ和HindⅢ双酶切后将其亚克隆入真核表达载体pcDNA3.1(-),并进行酶切及测序鉴定;将构建成功的pcDNA3.1(-)-pri-miR-7载体(命名为p-miR-7)体外瞬时转染95D细胞,应用Real-time PCR特异探针法检测miR-7成熟体的表达水平,并利用MTT法检测细胞生长的改变。结果酶切和测序验证成功构建了miR-7真核表达载体p-miR-7;表达载体p-miR-7瞬时转染95D细胞后可有效表达miRNA-7成熟体,并显著抑制95D细胞的体外生长(P<0.05)。结论成功构建了miR-7的真核表达载体,为后续深入研究miR-7在肺癌发生中的作用及机制提供了前期实验基础。

Objective To construct a eukaryotic expression vector encoding miR-7 and detect its effect on the growth of human lung cancer cells.Methods Pri-miR-7 sequence was amplified from genomic DNA of human 95D cells by PCR and subcloned into eukaryotic expression vector pcDNA3.1（-） using restriction site of BamHⅠ and HindⅢ.The positive recombinant pcDNA3.1（-）-pri-miR-7（p-miR-7） was identified by enzyme cleaving and sequencing.p-miR-7 was transiently transfected into 95D cells and the expression level of miRNA-7 was determined by Real-time PCR using specific probe before and after transfection.The proliferation of 95D cells was also accessed by MTT assay.Results Eukaryotic expression vector encoding miR-7（p-miR-7） was identified as correct by enzyme cleaving and sequencing.The expression level of miR-7 in 95D cells transiently transfected with p-miR-7 was increased significantly（P0.05）.The proliferation of 95D cells in vitro was markedly inhibited（P0.05）.Conclusion The eukaryotic expression vector pcDNA3.1（-）-pri-miR-7 is successfully constructed,which provides the experimental foundation for further studies on the role and mechanism of miR-7 in the pathogenesis of lung cancer.