Effect Of GSK-3β-targeting RNAi Combinant Adenovirus Vector On Proliferation Of Human Umbilical Vein Endothelial Cell

Objective:The aim of this project was to construct RNAi combinant adenoviral expressive vectors specific to GSK-3βto observe their gene silencing effect on GSK-3βgene, then initially approach the effect of Wnt/β-catenin pathway on proliferation of human umbilical vein endothelial cell using the RNAi adenovirus vector.Methods:The first step was to Design and synthesize three pairs of complementary single-strand DNA oligos that targeting three various sites of GSK-3βmRNA . Annealling was used to generate double-strand oligos(ds ligos),and then the ds oligos were cloned into pENTR^?/U6 to generate the Entry clone named pENTR. Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT^?-DEST was used to creat the adenovirus plasmid which contains the RNAi cassette. Then, transfected the adenovirus plasmids into HEK 293A cells to product adenovirus and amply the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. The GSK-3βgene silencing effect induced by the RNAi adenovirus was detected by westtern blot analysis and immunohistochemistry assay,also detectded its effect on the protein level ofβ-catenin. The expression of GSK-3βin HUVEC could be down-regulated by the RNAi adenovirus after infection, Wnt/β-catenin pathway could be influenced, then detedted the effect of Wnt/β-catenin pathway on proliferation of HUVEC with MTT assay.Results:The RNAi adenovirus specific to GSK-3βwere produced successfully with high titer .The expression of GSK-3βprotein in HUVEC could be down-regulated efficiently by the RNAi adenovirus,and the protein level ofβ-catenin could be increased obviously. The results of MTT assay suggested that interfering the GSK-3βwith the RNAi adenovirus might stimulate the proliferation of HUVEC.Conclusion:RNAi adenovirus is an important tool that can inhibit the expression of GSK-3βefficiently, hence influence the protein level ofβ-catenin obviously. Up-regulating of the Wnt/β-catenin pathway might play an important role on the proliferation of HUVEC.