Inhibitory effects of small interference RNA targeting vascular endothelial growth factor on oxygen-induced retinal neovascularization / 中华实验眼科杂志

ABSTRACT

Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.