Transcription factor SP1 mediates hyperglycemia-induced upregulation of roundabout4 in retinal microvascular endothelial cells
Transcription factor SP1 mediates hyperglycemia-induced upregulation of roundabout4 in retinal microvascular endothelial cells
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转录因子SP1介导高血糖诱导的视网膜微血管内皮细胞中roundabout4的上调
DOI：
10.1016/j.gene.2017.03.027
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发表时间：
2017-06-15
期刊：
影响因子：
3.5
通讯作者：
Su, Guanfang
中科院分区：
文献类型：
--
作者：
Xie, Jia'nan;Gong, Qiaoyun;Su, Guanfang
Roundabout 4（Robo 4）是一种在血管系统中特异性表达的基因，参与血管生成和血管完整性。随着糖尿病视网膜病变（DR）的发展，Robo 4的表达水平沿着逐渐升高。在本研究中，我们探讨了Robo 4在视网膜内皮细胞中的转录调控机制，并研究了这种调控对高血糖条件下细胞功能的影响。采用RT-qPCR和western blotting方法检测高糖刺激下人视网膜内皮细胞特异性蛋白1（SP1）和Robo 4的表达水平。利用小干扰RNA（SiRNA）转染技术分析SP1和Robo 4之间的调控关系。利用染色质免疫沉淀（ChIP）和荧光素酶分析研究转录因子SP1对Robo 4启动子活性的影响以及SP1结合位点的定位。进行细胞迁移、单层渗透性和管形成测定以证明SP1/Robo 4在高血糖条件下调节HREC功能的作用。结果表明，高血糖可上调HREC中SP1和Robo 4的mRNA和蛋白水平。通过siRNA转染消耗SP1抑制高血糖诱导的Robo 4过表达。ChIP结合荧光素酶分析表明，在高血糖条件下，SP1通过Robo 4启动子-1912/-1908处的额外SP1结合位点显著提高Robo 4的转录水平。抑制SP1/Robo 4通路可有效减轻高血糖对HREC迁移、通透性和血管生成的影响。所有这些发现表明，高血糖诱导的Robo 4上调是由SP1的转录增强介导的。SP1/Robo 4信号通路可调节高血糖条件下HREC的迁移能力、单层通透性和血管生成，提示其可能在DR过程中微血管功能障碍中发挥重要作用。（C）2017 Elsevier B. V.版权所有。
Roundabout4 (Robo4) is a gene that is expressed specifically in vasculature and is involved in the angiogenesis and integrity of blood vessels. The expression level of Robo4 increases gradually along with the development of diabetic retinopathy (DR). In this study, we explored the mechanism of transcriptional regulation of Robo4 in retinal endothelial cells, and investigated the effects of this regulation on cellular functions under hyperglycemic conditions. Human retinal endothelial cells (HREC) exposed to hyperglycemia were used to detect the expression levels of specificity protein 1 (SP1) and Robo4 by RT-qPCR and western blotting. Small interfering RNA (SiRNA) transfection technology was used to analyze the regulatory relationship between SP1 and Robo4. The effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation (ChIP) and luciferase assay. Cell migration, monolayer permeability and tube formation assays were performed to demonstrate the role of SP1/Robo4 in regulating HREC functions in hyperglycemic conditions. The results showed that hyperglycemia upregulated the mRNA and protein levels of SP1 and Robo4 in HREC. Depletion of SP1 by siRNA transfection inhibited the hyperglycemia induced overexpression of Robo4. ChIP combined with luciferase assay showed that under hyperglycemic conditions, SP1 significantly increased the transcriptional level of Robo4 via an additional SP1 binding site at -1912/-1908 in the Robo4 promoter. Repressing the SP1/Robo4 pathway effectively mitigated the abnormity in HREC migration, permeability and angiogenesis induced by hyperglycemia. All these findings indicate that hyperglycemia-induced upregulation of Robo4 is mediated by enhanced transcription of SP1. The SP1/Robo4 signaling pathway can regulate the migratory ability, monolayer permeability and angiogenesis of HREC under hyperglycemic conditions, suggesting that it may play an important role in microvascular dysfunction during DR. (C) 2017 Elsevier B.V. All rights reserved.