Construction of the recombinant adenovirus RNA interference vector of human hypoxia-inducible transcription factor-1? gene and its effect on expression of HIF-1? gene in human lung adenocarcinoma cells SPCA-1
- VernacularTitle:人HIF-1?基因RNAi重组腺病毒的构建及对人肺腺癌SPCA-1细胞HIF-1?表达的影响
- Author:
Xianrang SONG
;
Ling WEI
;
Xingwu WANG
;
Bao SONG
;
Yan ZHENG
- Publication Type:Journal Article
- Keywords:
lung neoplasm;
hypoxia-inducible transcription factor 1?;
RNA interference;
Adenovirus;
Gene therapy
- From:
Basic & Clinical Medicine
2006;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the RNA interference(RNAi)recombinant adenovirus vector targeting at human hypoxia-inducible transcription factor 1?(HIF-1?)and to evaluate its effect on human lung adenocarcinoma cell line SPCA-1.Methods The recombinant adenovirus Ad was constructed.HIF-1? inserted with HIF-1? 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.HIF1? in vitro,the expression rate of green fluorescence protein(GFP)was recorded by flow cytometry,HIF-1? mRNA and protein level was measured by Real-Time RT-PCR and flow cytometry.ResultsThe recombinant shuttle plasmid PAdTrack.HIF-1? and adenovirus plasmid Ad.HIF-1? were all correct shown by enzyme digestion confirmation.The plasmid pAd.HIF-1? 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-1? in vitro for 48 h,GFP expression rate was 92%,HIF-1? mRNA and protein level decreased 89% and 87%,respectively.Conclusion RNAi adenovirus vector of human HIF-1? gene has been successfully constructed,which could facilitate the research onHIF-1? gene related gene therapy for lung cancer.
