Construction of recombinant adenovirus vector encoding VEGF siRNA and its inhibitory effect on K562 cell proliferation

Objective To construct an recombinant adenovirus vector of vascular endothelial growth factor small interfering RNA(Ad5-VEGFsi) and to observe its inhibitory effect on the cell proliferation of human leukemia K562 cells. Methods The specific human VEGF siRNA was subcloned into the shuttle plasmid pSES-HUS and then cotransfected with plasmid pAdeasy-1 to produce pAd5-VEGFsi by homologous recombination. The identified recombinant plasmid pAd5-VEGFsi was packaged and amplified in 293 cells. At 72 h after the transfection of Ad5-VEGFsi into K562 cells, VEGF mRNA expression and the protein level of VEGF in the cell culture supernatant were determined by RT-PCR and ELISA, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by flow cytometry. Results The recombinant adenovirus Ad5-VEGFsi was successfully constructed. Ad5-VEGFsi with the titer of 4.6×1011 pfu/ml was harvested by CsCl gradient purification. Compared with those in the control, EGF mRNA in cells decreased by 66.55% (P0.01) but the VEGF protein concentration in the cell culture supernatant reduced by 86.03%(P0.01). As a consequence, the cell growth was inhibited, but the cell apoptosis rate increased 13.89% as compared with those in the control (P0.01). Conclusion Recombinant adenovirus Ad5-VEGFsi could effectively down-regulate VEGF expression in K562 cells, inhibit cell growth, and induce cell apoptosis.