The Construction Of Recombinant Over-expression Plasmid PcDNA3.1-HIF-1α Of Hypoxia-inducible Factor-1α And Its Effect And Significances In Prostate Cancer

Objective To construct recombinant over-expression plasmid pcDNA3.1-HIF-1α,and to discuss the effect and significances of HIF-1αin the occurrence and development of prostate cancer.Methods (1)According to gene bank, primer premier 5.0 was introduced to design the special primer of HIF-1α,which expanded the genenic segment of HIF-1αwith prostate cancer cDNA template by RT-PCR;(2)The augment segment of HIF-1αand plasmid pcDNA3.1 were digested with endonuclease BamH I and Xbar I,then to connnect;(3)The recombinant over-expression plasmid was transfected into prostate cancer PC-3,then to assay the expression of HIF-1αprotein by western blot;(4)Both the over-expression plasmid and RNA interference vector were transfencted into prostate cancer,then to select the stable expression cell line with G418 400ug/l about 4 weeks,the selected monoclone cells were continued to culture,and respectively named as pcDNA3.1-HIF-1α-PC-3,pcDNA3.1-PC-3,pll3.7-siHIF-1α-PC-3,pll3.7-PC-3 and PC-3,the expression of HIF-1α,VEGF,MMP-2,MMP-9 mRNA and protein were assayed in these cells by RT-PCR and western-blot, MTT and transwell chamber were adopted to describe proliferation and invasion of prostate cancer cell which was in the over-expression of HIF-1αResults The augment segment was the HIF-1αgenenic segment with electrophoresis and sequence identification,which was in accordance with the sequence published in the gene bank,and the recombinant plasmid can over-express in the transfected prostate cancer PC-3,showed that the recombinant plasmid of HIF-1α was constructed successfully;Both the pcDNA3.1-HIF-1α,RNA interference vector pll3.7-siHIF-1αwere transfected into logarithmic growth phase prostate cancer PC-3,the transfection rate were respectively (49.24±2.56)%,(42.36±2.62)%,the expression of HIF-1αmRNA was not increased in the over-expression cell compare to the other prostate cancer cells,but the protein was increased significantly,while both the mRNA and protein of HIF-1αwere decreased in the interference cell,the same as other factors associated with HIF-1αexcept for VEGF;The ability of proliferation was enhanced and the invasion cell number was increased in the over-expression by MTT and transwell chamber.Conclusion The recombinant plasmid of HIF-1αwas successfully constructed by RT-PCR,and the proliferation and invasion of prostate cancer were enhanced in the over-expression of HIF-1α,showed that HIF-1αhas close relationship with the development and invasion metastasis of prostate cancer,revealed that HIF-1αmay be an important factor in the pathogenesis of prostate cancer,which provide a new foundation for the pathogenesis research of prostate cancer,and genic therapy targeted at HIF-1αmay be a new track for the treatment of prostate cancer.