Lentivirus mediated small interference RNA targeting cyclic adenosine monophosphate responsive element binding protein 1 suppress retinal neovascularization in mice

Abstract

Objective To observe the inhibitory effect of lentivirus mediated small interference RNA (siRNA) targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) on retinal neovascularization (RNV) in mice.Methods CREB1 siRNA construct was created,screened and packaged to produce CREB1 RNAi-lentivirus.One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into 4 groups including normal group,oxygen induced retinopathy (OIR) group,empty vector group and CREB1 therapy group with 35 mice in each group.Mice in the normal group were kept in normal room air,while in the other three groups retinal neovascularization was induced by hypoxia on postnatal day 7 (P7).The mice in the OIR group were not treated.The mice in the vector group received intravitreal injection of lentivirus-green fluorescent protein (lenti-GFP,1 μl),and the CREB1 therapy group received CREB1 RNAi-lentivirus (1 μl) on P5.The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography.The areas of RNV and non-perfusion region were calculated.The expression of CREB1,phosphorylated-CREB1 (P-CREB1) and vascular endothelial growth factor (VEGF)-A levels,Akt and phosphoinositide 3-kinases (PI3K) in retinas were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot.Results The number of vascular cell nuclei breaking through the ILM of the OIR group and the empty vector group increased significantly compared with the normal group (P＜0.05),while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group(P＜0.05).The area of RNV and non-perfusion region of the OIR group and the empty vector group increased significantly compared with the normal group,while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group.The difference of area of RNV and non-perfusion region among 4 groups were significant (F=67.220,110.090; P＜0.05).The mRNA expression of CREB1 and protein expression of P-CREB1,the mRNA and protein expression of VEGF-A,Akt,PI3K in the retina were increased significantly in the OIR group and the empty vector group as compared with the normal group,while decreased significantly in the CREB1 therapy group as compared with the OIR group and the empty vector group.The difference of mRNA expression of CREB1,VEGF-A,Akt,PI3K in the retina among 4 groups were significant (F=6.087,5.464,6.191,8.627; P＜0.05).The protein expression of P-CREB1,VEGF-A,Akt,PI3K in the retina among 4 groups were significant (F=162.944,13.861,19.710,22.827; P＜0.05).Conclusion RNV in the mice is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1 down-regulation. Key words: Retinal neovascularization/prevention & control; Gene transfer techniques; Transfection; Animal experimentation