Lo Wan-Yu, Wang Shou-Jie, Wang Huang-Joe
Cardiovascular and Translational Medicine Laboratory, Department of Biotechnology, Hungkuang University, Taichung, Taiwan.
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
Front Physiol. 2020 Oct 30;11:1091. doi: 10.3389/fphys.2020.01091. eCollection 2020.
Increased -GlcNAc transferase (OGT)-induced O-linked -acetylglucosamine (-GlcNAc) post-translational modification is linked with diabetic complications. MicroRNA-146a-5p (miR-146a-5p) is a negative inflammatory regulator and is downregulated in diabetes. Here, we investigated the interaction between miR-146a-5p and OGT. Human aortic endothelial cells (HAECs) were stimulated with high glucose (25 mM) and glucosamine (25 mM) for 24 h. Western blot, real time PCR, bioinformatics analysis, luciferase reporter assay, miR-146a-5p mimic/inhibitor transfection, siRNA OGT transfection, miR-200a/200b mimic transfection, and OGT pharmacological inhibition (ST045849) were performed. The aorta from miR-146a-5p mimic-treated db/db mice were examined by immunohistochemistry staining. HG and glucosamine upregulated OGT mRNA and protein expression, protein -GlcNAcylation, and IL-6 mRNA and protein expression. Real time PCR analysis found that miR-146a-5p was decreased in HG- and glucosamine-stimulated HAECs. This suggested that OGT-induced protein O-GlcNAcylation as a mechanism to downregulate miR-146a-5p. Bioinformatic miR target analysis excluded miR-146a-5p as a post-transcriptional regulator of OGT. However, a luciferase reporter assay confirmed that miR-146a-5p mimic bound to 3'-UTR of human OGT mRNA, indicating that OGT is a non-canonical target of miR-146a-5p. Transfection with miR-146a-5p mimic and inhibitor confirmed that miR-146a-5p regulated OGT/protein O-GlcNAcylation/IL-6 expression levels. Furthermore, OGT siRNA transfection, miR-200a/miR-200b mimic transfection, and ST045849 increased HG-induced miR-146a-5p expression levels, indicating that HG-induced miR-146a-5p downregulation is partially mediated through OGT-mediated protein -GlcNAcylation. In , intravenous injections of miR-146a mimic decreased endothelial OGT and IL6 expression in db/db mice. A non-canonical positive feedback interaction between miR-146a-5p and OGT is involved in a vicious cycle to aggravate HG-induced vascular complications.
增强的O-连接N-乙酰葡糖胺转移酶（OGT）诱导的O-连接β-N-乙酰葡糖胺（O-GlcNAc）翻译后修饰与糖尿病并发症相关。微小RNA-146a-5p（miR-146a-5p）是一种负性炎症调节因子，在糖尿病中表达下调。在此，我们研究了miR-146a-5p与OGT之间的相互作用。用人主动脉内皮细胞（HAECs）在高糖（25 mM）和葡糖胺（25 mM）条件下刺激24小时。进行了蛋白质印迹、实时定量PCR、生物信息学分析、荧光素酶报告基因检测、miR-146a-5p模拟物/抑制剂转染、OGT小干扰RNA转染（siRNA OGT）、miR-200a/200b模拟物转染以及OGT药理学抑制（ST045849）实验。通过免疫组织化学染色检测了用miR-146a-5p模拟物处理的db/db小鼠的主动脉。高糖和葡糖胺上调了OGT的mRNA和蛋白表达、蛋白质O-GlcNAc化以及白细胞介素-6（IL-6）的mRNA和蛋白表达。实时定量PCR分析发现，在高糖和葡糖胺刺激的HAECs中miR-146a-5p表达降低。这表明OGT诱导的蛋白质O-GlcNAc化是下调miR-146a-5p的一种机制。生物信息学miR靶标分析排除了miR-146a-5p作为OGT转录后调节因子的可能性。然而，荧光素酶报告基因检测证实miR-146a-5p模拟物与人OGT mRNA的3'-非翻译区（3'-UTR）结合，表明OGT是miR-146a-5p的非经典靶标。用miR-146a-5p模拟物和抑制剂转染证实miR-146a-5p调节OGT/蛋白质O-GlcNAc化/IL-6的表达水平。此外，OGT siRNA转染、miR-200a/miR-200b模拟物转染以及ST045849增加了高糖诱导的miR-146a-5p表达水平，表明高糖诱导的miR-146a-5p下调部分是通过OGT介导的蛋白质O-GlcNAc化实现的。在db/db小鼠中，静脉注射miR-146a模拟物可降低内皮细胞中OGT和IL-6的表达。miR-146a-5p与OGT之间的非经典正反馈相互作用参与了一个恶性循环，加剧了高糖诱导的血管并发症。
