VEGF Short Hairpin RNA Expression Plasmid Blocks VEGF Expression In Vascular Endothelial Cells And Inhibits Rat Corneal Neovascularization

Objective: To determine whether RNA interference could block vascular endothelial growth factor (VEGF) in vascular endothelial cells in vitro and inhibit cautery-induced rat corneal neovascularization in vivo. Methods: Three shRNAs targeting VEGF mRNA were designed and synthesized, VEGF shRNA expression plasmids were constructed using gene engineering technique. Then the recombinant plasmids were transfected into vascular endothelial cells with Lipofectamine 2000 transfection system. Expression of VEGF mRNA and protein in transfected cells was detected by RT-PCR and Western blot respectively. Corneal neovascularization in rat eyes was induced by alkaline cautery, then the study eyes were received subconjunctival injection of VEGF shRNA3 plasmid. The corneal neovessels were evaluated with microscope. After seven days, expression of VEGF mRNA and protein was detected by RT-PCR and Western blot respectively. Results: VEGF shRNA expression plasmids were successfully constructed and identified by restriction enzyme and sequence analysis. Expression of VEGF mRNA and protein was down regulated by VEGF shRNA1, VEGF shRNA2 and VEGF shRNA3 plasmids in ECs, and VEGF shRNA3 plasmid showed stronger inhibitory effect, the inhibitory effects of VEGF mRNA and protein were 68.92% and 66.22%. Subconjunctival injection of VEGF shRNA3 plasmid reduced rats′corneal neovessels compared with controls. Expression of VEGF mRNA and protein was down regulated by VEGF shRNA3 plasmid in rats′corneas, the inhibitory effects of VEGF mRNA and protein were 41.84% and 41.86%. Conclusions: VEGF shRNA expression plasmids significantly suppressed expression of VEGF in vascular endothelial cells in vitro. In vivo, subconjunctival delivery of VEGF shRNA3 plasmid suppressed cautery-induced corneal neovascularization and VEGF expression.