Construction of pcDNA3-HIF-1? eukaryotic expression vector and its expression in MCF-7 cells / 基础医学与临床

ABSTRACT

Objective To construct pcDNA3-HIF-1? eukaryotic expression vector and to investigate its function in breast cancer MCF-7 cells.Methods RT-PCR was applied to amplify human HIF-1? cDNA from MCF-7 cells with a pair of sequence specific primers carrying a restriction enzyme site XbaⅠ or HindⅢ on each 5′end.HIF-1?(cDNA) was inserted into pMD18-T after sequencing,and then inserted into pcDNA3 vector.The recombinant vector was transfected into MCF-7 cells to observe its expression and function by Western blot and dual-luciferase reporter gene assay.Realtime-PCR was performed to detect the inducible expression of C-MET mRNA by HIF-1?,the anti-apoptotic effect of HIF-1 under hypoxia was analysed by detecting the Caspase3/7 activity.Results The sequence of pcDNA3-HIF-1? is identical with the gene bank.It enhances the expression of HRE reporter gene and C-MET mRNA,but decreases the Caspase3/7 activity in MCF-7 cells under hypoxia.Conclusion The pcDNA3-HIF-1? eukaryotic expression vector was successfully constructed,it not only has strong DNA binding and inducing activity,but also has anti-apoptotic effect in hypoxia MCF-7 cells.