Construction and identification of eukaryotic expression vector encoding miRNA-7

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.