Inhibitory effects of chemically synthetic small interference RNA on hypoxia-inducible factor-1α expression in rat retinal vascular endothelial cells of hypoxic condition

Objective To investigate the effects of hypoxia-inducible factor-1α (HIF-1α)expression on pathogenesis of retinopathy of prematurity (ROP) and to find new target for gene therapy.Methods After liposome-mediated small interference RNA (siRNA) transfection into rat retinal endothelial cells,the cells were cultured in medium with CoCl2-induced hypoxic condition.Expression of HIF-1α mRNA was determined by fluorenscence quantitative reverse transcription-polymerase chain reaction(RT-PCR),HIF-1α protein expression was detected by Western Blot after cocultured for 8 hours.Cell proliferation was measured with 3-(4,5)-dimethylthiazol (-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay after cocultured for 24 hours.Difference between groups was compared with independent samples t test.Results Rat retinal vascular endothelial cells were successfully transfected with siRNA.Fluorescence quantitative RT-PCR results showed that at 48 hours of transfection,the expression of HIF-1α mRNA in the interference group of siRNA1,siRNA2 and siRNA4 were 0.1620 ± 0.0147,0.2034 ± 0.0251 and 0.3049 ± 0.0165,which were 16.20％,20.34 ％ and 30.49％ of blank control group (1.0000±0.0344),and were lower than that of negative control group (0.8334±0.0242) (t=16.786,8.953 and 4.087,P＜0.05 respectively).Western Blot results showed that HIF-1α protein expression was significantly inhibited by siRNA1(0.4956 ± 0.0421 ) and siRNA2 (0.6544 ± 1.0032) comparing with blank control group (3.5105 ±0.4084) and negative control group (3.4019 ± 1.0677) (t =6.861,2.893,4.567 and 5.072,P＜0.05 respectively).As for cellular proliferation activity,(49.5±2.9) ％ and (67.4±1.2) ％ of cells growth inhibition were observed after transfection with siRNA1 and siRNA2,which were higher than those of negative control group [(15.7±1.5) ％ ] (t=2.786 and 6.904,P＜0.05).Conclusions The synthetic HIF-1α siRNA could effectively inhibit the expression of HIF-1α gene and reduce cell proliferation in rat retinal endothelial cells under hypoxic condition.RNA interference technology targeting HIF-1α might become a new strategy for gene therapy of ROP. Key words: RNA,small interfering; Hypoxia-inducible factor 1,alpha subunit; Anoxia; Retinal vessels; Endothelial cells; Cell proliferation