The inhibition effect of interfering RNA target HIF-1¦Á on VEGF mRNA expression in the retina of diabetic rats

Song Chen, Li-Zhu Meng

Clinical college of ophthalmology, Tianjin Medical University, Tianjin Eye Hospital, Tianjin institute of ophthalmology , Tianjin 300020, China

Objective: To evaluate the inhibitory effect of the hypoxia inducible factor-1¦Á£¨HIF-1¦Á£© specific siRNA on the expression of vascular endothelial growth factor (VEGF)mRNA in retina issues in diabetic rat.

Methods: Randomized controlled trail was performed. Using pSilencer2.1-U6neo for plasmid vector, HIF-1¦Á specific siRNA recombinant plasmid was constructed .There was totally fifty-four healthy Sprague Dawley rats in which fifteen rats were chosen as normal group and thirty-nine rats were constructed for diabetic retinopathy model by streptozotocin (STZ) which was divided into three groups randomly including control model group (DR group) ,vector group and gene therapy group(HIF-1¦ÁsiRNA group). Each group contained twelve rats.The rest were used for stretched preparation. FITC-Dextran fluorescent angiography was used to assess the capillary vessel pattern to ensure modeling construction succeed after 18 weeks. LipofectamineTM2000 with pSilencer vector plasmid£¬HiF-1¦Á siRNA were injected into the vitreous in the vector group and HiF-1¦ÁsiRNA group respectively. Nothing was transfected into DR group and normal group.The expression of VEGF mRNA in retinas was measured by Real-time reverse transcriptase-polymerase chain reaction (RT-PCR).Significant differences between groups were evaluated by one-way analysis of variance,followed by LSD-t analysis.

Results: HIF-1¦Á siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. FITC-Dextran fluorescent angiography proved modeling construction successfully. String-of-beads , fluorescein leakage and large nonperfusioncan be found in retinas of diabeitc rats.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the normal group, but increased obviously in the DR group(P<0.05).There was no statistically significant between DR group and vector group(P>0.05). The expression of VEGFmRNA in HIF-1¦Á siRNA group were obviously decreased compared with DR group. VEGFmRNA level was reduced by 32.76%¡¢43.60%¡¢47.70%¡¢50.86% at 24h¡¢48h¡¢72h¡¢1w.

Conclusions: VEGFmRNA can be efficiently inhibited by LipofectamineTM2000 with HIF-1¦Á siRNA through intravitreal injection in the diabetic rats. New gene therapy may provided to control new vessels of diabetic retinopathy.

Keywords: Hypoxia inducible factor(HIF), Vascular endothelial growth factor(VEGF), RNA interfering, Retinal neovascular,Gene therapy