miR-20a-5p调节VEGF通路在氧诱导视网膜病变小鼠模型中的作用机制

The mechanism of miR-20a-5p regulating VEGF pathway in the mouse model of retinopathy of prematurity

目的探讨微小RNA(miR)-20a-5p调节血管内皮生长因子(VEGF)通路在氧诱导视网膜病变(OIR)小鼠中的作用机制。方法实验分为正常组、模型组、高氧对照组、miR-20a-5p高表达组,每组24只。除正常组外,其余组小鼠在出生后第7天置于氧浓度(75.00±2.00)%氧箱中建立OIR小鼠模型,连续高氧环境5 d后置于常氧中饲养。高氧环境结束前1 d,高氧对照组玻璃体腔内注射1μL磷酸缓冲盐溶液(PBS),miR-20a-5p高表达组玻璃体腔内注射1μL miR-20a-5p激动剂(miR-20a-5p agomir)(1μmol/L),模型组不进行任何处理。正常组一直置于空气中正常饲养。高氧结束后各组小鼠正常空气再饲养5 d进行实验。实时荧光定量PCR(qRT-PCR)检测视网膜组织miR-20a-5p、VEGF、血管内皮生长因子受体(VEGFR)-1、VEGFR-2表达情况;视网膜铺片观察视网膜血管形态;苏木精-伊红(HE)染色计数视网膜新生血管内皮细胞核情况;免疫组化检测视网膜组织中VEGF阳性细胞情况。结果模型组和高氧对照组在出生第17天自视盘向周围发出的放射状大血管迂回、不规则扩张,出现大量新生血管,且新生血管结构及分布紊乱,周边毛细血管网闭塞;miR-20a-5p高表达组相较模型组和高氧对照组在出生第17天自视盘向周围发出的放射状大血管迂回不明显,血管不规则扩张现象减轻,新生血管明显减少。与正常组相比,模型组、高氧对照组视网膜组织中miR-20a-5p水平降低(P<0.05),视网膜血管内皮细胞核数量、VEGF蛋白阳性面积百分比、视网膜组织中VEGF、VEGFR-1、VEGFR-2 mRNA表达水平升高(P<0.05);分别与模型组、高氧对照组相比,miR-20a-5p高表达组视网膜组织中miR-20a-5p水平升高(P<0.05),视网膜血管内皮细胞核数量、VEGF蛋白阳性面积百分比、视网膜组织中VEGF、VEGFR-1、VEGFR-2mRNA表达水平降低(P <0.05)。结论升高miR-20a-5p可抑制VEGF通路从而减少OIR小鼠视网膜血管新生,实现对OIR小鼠视网膜的保护。

Objective To investigate the role of microRNA(miR)-20a-5p in the regulation of the vascular endothelial growth factor(VEGF)pathway in mice with oxygen-induced retinopathy(OIR).Methods Mice were divided into a normal group,model group,hyperoxia control group,and miR-20a-5p high expression group,with 24 mice in each group.With the exception of the normal group,the mice were placed in an oxygen tank with(75.00±2.00)%oxygen concentration beginning postnatal day 7 to establish the OIR model,and after 5 days of continuous hyperoxia environment,they were returned to normal air conditions.At 1 day before the end of the hyperoxia environment,1μL phosphate buffered saline was injected into the vitreous cavity of the hyperoxia control group,1μL miR-20a-5p agomir(1μmol/L)was injected into the vitreous cavity of the high expression group,and the model group received nothing.The normal group was kept in normal air conditions.The experiment was carried out after the mice were exposed to normal air for 5 more days.The expressions of miR-20a-5p,VEGF,VEGF receptor(VEGFR)-1 and VEGFR-2 were detected by real-time fluorescence quantitative PCR,retinal vascular morphology was observed by retinal patch,hematoxylin and eosin staining was used to count the endothelial cells of retinal neovascularization,and VEGF-positive cells were detected by immunohistochemistry.Results In the model group and hyperoxia control group,on postnatal day 17,the large radial blood vessels extending from the optic disc were round and irregular.There were many new blood vessels;the structure and distribution of neovascularization were disordered and the capillary network was occluded.Compared with the model group and the hyperoxia control group,the miR-20a-5p high expression group had no obvious circuitous radial vasculature and had less irregular vascular expansion and neovascularization.Compared with those in the normal group,the retinal miR-20a-5p levels in the model group and hyperoxia control group were lower(P<0.05),and the number of nuclei in retinal vascular endothelium,VEGF protein-positive area percentage,and expressions of VEGF,VEGFR-1 and VEGFR-2 mRNAs in retina were higher(P<0.05).Compared with those in the model group and the hyperoxia control group,the retinal miR-20a-5p level in the miR-20a-5p high expression group was lower(P<0.05),and the number of nuclei in retinal vascular endothelium,VEGF protein-positive area percentage,and expressions of VEGF,VEGFR-1 and VEGFR-2 mRNAs in retina were higher(P<0.05).Conclusions Increasing miR-20a-5p inhibited the VEGF pathway and decreased retinal neovascularization in OIR mice,which may protect the retina of OIR mice.