miR-106b对缺氧诱导的大鼠视网膜血管化的影响及其机制

Effect and mechanism of micro ribonucleic acid-106b on hypoxia-induced rat retinal vascularization

目的探讨miR-106b对缺氧诱导的大鼠视网膜血管化的影响及其机制。方法本研究实验动物为无特定病原菌级SD怀孕大鼠所产的12只新生大鼠。将新生大鼠与母鼠(用于哺乳)随机分配至对照组与氧诱导视网膜病变(OIR)组,每组6只,对照组正常饲养,OIR组新生大鼠建立OIR模型。采用免疫荧光染色观察大鼠视网膜的组织学变化,采用实时荧光定量PCR检测大鼠视网膜组织中miR-106b的表达情况。分离大鼠视网膜微血管内皮细胞(MRMVEC),采用双荧光素酶报告基因系统实验与Western blot验证miR-106b与缺氧诱导因子-1A(HIF1A)、血管内皮生长因子A(VEGFA)的靶向关系。将MRMVEC分为5组:Nor组(常氧培养)、Hyp组(缺氧处理)、Hyp-miR组(转染miR-106b mimic后接受缺氧处理)、Hyp-miR-pcHIF组(转染pcDNA-HIF1A与miR-106b mimic后接受缺氧处理)与Hyp-miR-pcVEG组(转染pcDNA-VEGFA与miR-106b mimic后接受缺氧处理);分别采用CCK-8法、划痕实验与成管实验检测细胞增殖、迁移及体外血管形成能力。结果免疫荧光染色结果显示:与对照组相比,OIR组大鼠视网膜血管内皮细胞增殖与迁移明显增强。实时荧光定量PCR检测结果显示:OIR组大鼠视网膜组织中miR-106b相对表达量(0.26±0.06)显著低于对照组(1.02±0.08),差异有统计学意义(P<0.05)。双荧光素酶报告基因系统实验与Western blot结果表明,miR-106可与HIF1A和VEGFA靶向结合并抑制其表达。与Nor组相比,Hyp组MRMVEC的相对细胞活力、相对迁移能力、相对血管形成能力均提高,差异均有统计学意义(均为P<0.05);与Hyp组相比,Hyp-miR组MRMVEC的相对细胞活力、相对迁移能力、相对血管形成能力均降低,差异均有统计学意义(均为P<0.05);与Hyp-miR组相比,Hyp-miR-pcHIF组与Hyp-miR-pcVEG组MRMVEC的相对细胞活力、相对迁移能力、相对血管形成能力均有所提高,差异均有统计学意义(均为P<0.05)。结论上调miR-106b可靶向抑制HIF1A和VEGFA表达,从而减少缺氧诱导的视网膜微血管内皮细胞增殖、迁移与新生血管生成。

Objective To investigate the effect of micro ribonucleic acid(miR)-106b on hypoxia-induced retinal neovascularization and its mechanism.Methods Totally 12 neonatal rats born of specific-pathogen-free Sprague-Dawley rats were selected for the study.Neonatal rats and mother rats(for lactation)were randomly assigned to a control group and an oxygen-induced retinopathy(OIR)group,with 6 rats in each group.Rats in the control group were fed normally,and OIR models were established in newborn rats in the OIR group.The histological changes of the retina were observed by immunofluorescence staining,and the expression of miR-106b in retina tissues was detected by real-time fluorescence quantitative PCR.The retinal microvascular endothelial cells(RMVEC)of rats were isolated.The targeted relationship between miR-106b and hypoxia-inducible factor-1A(HIF1A)and vascular endothelial growth factor A(VEGFA)was verified by dual-luciferase reporter assay and Western blot.RMVEC were divided into 5 groups:Nor group(normoxic culture),Hyp group(hypoxia treatment),Hyp-miR group(hypoxia treatment after transfection with miR-106b mimic),Hyp-miR-pcHIF group(hypoxia treatment after transfection with pcDNA-HIF1A and miR-106b mimic)and Hyp-miR-pcVEG group(hypoxia treatment after transfection with pcDNA-VEGFA and miR-106b mimic).Cell proliferation,migration and in-vitro angiogenesis capacity were detected by Cell Counting Kit-8,scratch test and tube formation assay,respectively.Results The immunofluorescence staining showed that compared with the control group,the proliferation and migration of retinal vascular endothelial cells in the OIR group significantly increased.The real-time fluorescence quantitative PCR detection results showed that the relative expression of miR-106b in the retina tissues of rats in the OIR group(0.26±0.06)was significantly lower than that of the control group(1.02±0.08)(P<0.05).The results of dual-luciferase reporter assay and Western blot showed that miR-106b could realize a targeted binding with HIF1A and VEGFA,and reduce their expressions.Compared with the Nor group,the relative cell viability,relative migration ability,and relative angiogenesis ability of RMVEC in the Hyp group improved,with statistically significant differences(all P<0.05);compared with the Hyp group,the relative cell viability,relative migration ability and relative angiogenesis ability of RMVEC in the Hyp-miR group decreased,with statistically significant differences(all P<0.05);compared with the Hyp-miR group,these parameters of RMVEC in the Hyp-miR-pcHIF group and Hyp-miR-pcVEG group improved,with statistically significant differences(all P<0.05).Conclusion Upregulation of miR-106b can targetedly inhibit the expressions of HIF1A and VEGFA,thereby reducing hypoxia-induced proliferation,migration and neovascularization of RMVEC.