Construction of the recombinant adenovirus RNA interference vector of human hypoxia-inducible transcription factor-1α gene and its effect on expression of HIF-1α gene in human lung adenocarcinoma cells SPCA-1

Objective To construct the RNA interference(RNAi)recombinant adenovirus vector targeting at human hypoxia-inducible transcription factor 1α(HIF-1α)and to evaluate its effect on human lung adenocarcinoma cell line SPCA-1.Methods The recombinant adenovirus Ad was constructed.HIF-1α inserted with HIF-1α RNAi fragment via AdEasy system.The virus was purifed by CsCl gradient centrifuge.The functional titer of recombinant adenovirus was measured by transfection test in HEK 293 cells.SPCA-1 cells were transducted with 2 multiplicity of infection(MOI)Ad.HIF1α in vitro,the expression rate of green fluorescence protein(GFP)was recorded by flow cytometry,HIF-1α mRNA and protein level was measured by Real-Time RT-PCR and flow cytometry.ResultsThe recombinant shuttle plasmid PAdTrack.HIF-1α and adenovirus plasmid Ad.HIF-1α were all correct shown by enzyme digestion confirmation.The plasmid pAd.HIF-1α was transducted into HEK293 cells,15%GFP expressionwere seen after 3 days.The final titers of recombinant adenovirus were 5.0×1010 TU/mL.SPCA-1 cells was transducted by Ad.HIF-1α in vitro for 48 h,GFP expression rate was 92%,HIF-1α mRNA and protein level decreased 89% and 87%,respectively.Conclusion RNAi adenovirus vector of human HIF-1α gene has been successfully constructed,which could facilitate the research onHIF-1α gene related gene therapy for lung cancer.