Zhang Lixin, Tao Lijuan
Department of Ophthalmology, Hunan Children's Hospital Changsha 410008, Hunan, P. R. China.
Int J Clin Exp Pathol. 2017 Aug 1;10(8):8845-8857. eCollection 2017.
Retinal neovascularization (RNV) is a prominent pathological angiogenesis, which causes detrimental outcomes in visual functions. Previous literature represents that miR-132 induces angiogenesis in tumor development and ischemic diseases. Considering the important role in angiogenesis, we hypothesized that miR-132 might be involved in RNV. In this study, human retinal microvascular endothelial cells were maintained in hypoxia for indicated time, followed by further incubation in normoxic conditions to establish hypoxia/reoxygenation (H/R) models . mRNA microarray analysis was undertaken to detect alterations in gene profiles in the cells. qRT-PCR and Western blotting were performed to evaluate expression of genes that are closely associated to neovascularization. Results showed that miR-132 expression was increased under hypoxic conditions. Reoxygenation for a limited time (6 h) failed to restore miR-132 expression to basal level. Interference of miR-132 expression via its inhibitor suppressed the cell proliferation under H/R conditions, increasing the apoptosis rate. mRNA microarray analysis revealed that miR-132 is involved in the regulation of vasculature development, blood vessel morphogenesis, and proliferation and migration of microvascular endothelial cells through regulating genes such as early growth response gene 1 (Egr1), extracellular signal-regulated kinase (ERK), metal matrix proteinase (MMP2), vascular endothelial growth factor (VEGF)-A and VEGF-C. qRT-PCR and Western blotting further demonstrated that miR-132 up-regulated their gene and protein expression under H/R conditions. In summary, miR-132 was involved in the development of RNV under H/R conditions, at least partly, through up-regulating Egr1, ERK2, MMP2, VEGFA and VEGFC expression. This finding facilitates the understanding of pathogenic mechanisms of RNV.
视网膜新生血管形成（RNV）是一种显著的病理性血管生成，会导致视觉功能出现有害后果。既往文献表明，miR-132在肿瘤发展和缺血性疾病中诱导血管生成。鉴于其在血管生成中的重要作用，我们推测miR-132可能参与了视网膜新生血管形成。在本研究中，将人视网膜微血管内皮细胞在缺氧条件下培养指定时间，随后在常氧条件下进一步孵育以建立缺氧/复氧（H/R）模型。进行mRNA微阵列分析以检测细胞中基因谱的变化。采用qRT-PCR和蛋白质免疫印迹法评估与新生血管形成密切相关的基因表达。结果显示，在缺氧条件下miR-132表达增加。限时复氧（6小时）未能将miR-132表达恢复至基础水平。通过其抑制剂干扰miR-132表达可抑制H/R条件下的细胞增殖，增加凋亡率。mRNA微阵列分析显示，miR-132通过调节早期生长反应基因1（Egr1）、细胞外信号调节激酶（ERK）、金属基质蛋白酶（MMP2）、血管内皮生长因子（VEGF）-A和VEGF-C等基因，参与血管系统发育、血管形态发生以及微血管内皮细胞的增殖和迁移的调控。qRT-PCR和蛋白质免疫印迹法进一步证明，在H/R条件下miR-132上调了它们的基因和蛋白表达。总之，miR-132至少部分地通过上调Egr1、ERK2、MMP2、VEGFA和VEGFC的表达，参与了H/R条件下视网膜新生血管形成的发展。这一发现有助于理解视网膜新生血管形成的致病机制。
