AUTHOR=Lo Wan-Yu , Wang Shou-Jie , Wang Huang-Joe 

TITLE=Non-canonical Interaction Between O-Linked N-Acetylglucosamine Transferase and miR-146a-5p Aggravates High Glucose-Induced Endothelial Inflammation

JOURNAL=Frontiers in Physiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2020.01091

DOI=10.3389/fphys.2020.01091

ISSN=1664-042X

ABSTRACT=<p><bold>Background and Aims:</bold> Increased <italic>O</italic>-GlcNAc transferase (OGT)–induced O-linked <italic>N</italic>-acetylglucosamine (<italic>O</italic>-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.</p><p><bold>Methods:</bold> 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.</p><p><bold>Results:</bold> HG and glucosamine upregulated OGT mRNA and protein expression, protein <italic>O</italic>-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 <italic>O</italic>-GlcNAcylation. In <italic>vivo</italic>, intravenous injections of miR-146a mimic decreased endothelial OGT and IL6 expression in db/db mice.</p><p><bold>Conclusion:</bold> A non-canonical positive feedback interaction between miR-146a-5p and OGT is involved in a vicious cycle to aggravate HG-induced vascular complications.</p>