Tang Song-Tao, Wang Feng, Shao Min, Wang Yuan, Zhu Hua-Qing
Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, China; Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Department of Ophthalmology, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Vascul Pharmacol. 2017 Jan;88:48-55. doi: 10.1016/j.vph.2016.12.002. Epub 2016 Dec 18.
MicroRNA-126(miR-126) targets involved in inflammation need to be identified. In this study, we aim to investigate whether high-mobility group box 1(HMGB1), an inflammation-related gene, is the target of miR-126 in diabetic vascular endothelium. The diabetic apoE mice model, a classical diabetic atherosclerosis model, was established. The aorta of diabetic apoE mice showed decrease of miR-126 and elevation of HMGB1 and inflammation. Next, we employed several in vitro experiments to address the role of miRNA-126 on the regulation of HMGB1 in endothelial cells under hyperglycemic and inflammatory conditions. Manipulation of miRNA levels in human umbilical vein endothelial cells (HUVECs) was achieved by transfecting cells with miR-126 mimic and antagomir. Overexpression of miR-126 could decrease the expression of downstream components of HMGB1 including TNF-α, ROS, and NADPH oxidase activity in HUVECs under hyperglycemic condition. Nevertheless, such phenomenon was completely reversed by miR-126 antagomir. The expression of HMGB1 protein rather than HMGB1 mRNA was down-regulated after transfection with miR-126 mimic, which indicated the modulation of HMGB1 mediated by miR-126 was at the posttranslational level. Luciferase reporter assay confirmed the 3'-UTR of HMGB1 gene was a direct target of miR-126. Western blot analysis also indicated that overexpression of miR-126 contributed to the elevation of p-eNOS, eNOS and p-AKT expressions, respectively. In summary, our findings suggest that miR-126 may suppress inflammation and ROS production in endothelial cells treated by high glucose through modulating the expression of HMGB1. Our study provides a novel pathogenic link between dysregulated miRNA expression and inflammation in diabetic vascular endothelium.
需要鉴定参与炎症反应的微小RNA-126（miR-126）的靶标。在本研究中，我们旨在探究炎症相关基因高迁移率族蛋白B1（HMGB1）是否为糖尿病血管内皮中miR-126的靶标。建立了糖尿病载脂蛋白E小鼠模型，这是一种经典的糖尿病动脉粥样硬化模型。糖尿病载脂蛋白E小鼠的主动脉显示miR-126水平降低，HMGB1水平升高且伴有炎症反应。接下来，我们进行了多项体外实验，以研究在高血糖和炎症条件下，微小RNA-126对内皮细胞中HMGB1调控的作用。通过用miR-126模拟物和拮抗剂转染人脐静脉内皮细胞（HUVECs）来调控其微小RNA水平。在高血糖条件下，miR-126的过表达可降低HUVECs中HMGB1下游成分的表达，包括肿瘤坏死因子-α（TNF-α）、活性氧（ROS）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶活性。然而，miR-126拮抗剂可完全逆转这种现象。用miR-126模拟物转染后，HMGB1蛋白而非HMGB1 mRNA的表达下调，这表明miR-126介导的对HMGB1的调控发生在翻译后水平。荧光素酶报告基因检测证实HMGB1基因的3'-非翻译区（3'-UTR）是miR-126的直接靶标。蛋白质免疫印迹分析还表明，miR-126的过表达分别导致磷酸化内皮型一氧化氮合酶（p-eNOS）、内皮型一氧化氮合酶（eNOS）和磷酸化蛋白激酶B（p-AKT）表达升高。总之，我们的研究结果表明，miR-126可能通过调节HMGB1的表达来抑制高糖处理的内皮细胞中的炎症反应和ROS生成。我们的研究提供了糖尿病血管内皮中微小RNA表达失调与炎症之间一种新的致病联系。
