Dataset Information

MicroRNA-146a-5p Mediates High Glucose-Induced Endothelial Inflammation via Targeting Interleukin-1 Receptor-Associated Kinase 1 Expression.

ABSTRACT: Background and Aims: Interleukin-1 receptor-associated kinase-1 (IRAK-1) is critical for mediating toll-like receptor and interleukin-1 receptor signaling. In this study, we have examined whether IRAK-1 expression is altered in high glucose (HG)-stimulated human aortic endothelial cells (HAECs), and whether microRNAs (miRs) target IRAK-1 to regulate HG-induced endothelial inflammation. Methods: HAECs were treated with HG for 24 and 48 h. Real-time PCR, Western blot, monocyte adhesion assay, bioinformatics analysis, TaqMan® arrays, microRNA mimic or inhibitor transfection, luciferase reporter assay and siRNA IRAK-1 transfection were performed. The aortic tissues from db/db type 2 diabetic mice were examined by immunohistochemistry staining. Results: HG time-dependently increased IRAK-1 mRNA and protein levels in HAECs, and was associated with increased VCAM-1/ICAM-1 gene expression and monocyte adhesion. Bioinformatic analysis, TaqMan® arrays, and real-time PCR were used to confirm that miR-146a-5p, miR-339-5p, and miR-874-3p were significantly downregulated in HG-stimulated HAECs, suggesting impaired feedback restraints on HG-induced endothelial inflammation via IRAK-1. However, only miR-146a-5p mimic transfection reduced the HG-induced upregulation of IRAK-1 expression, VCAM-1/ICAM-1 expression, and monocyte adhesion. Additionally, IRAK-1 depletion reduced HG-induced VCAM-1/ICAM-1 gene expression, and monocyte adhesion, indicating that HG-induced endothelial inflammation was mediated partially through IRAK-1. In vivo, intravenous injections of miR-146a-5p mimic prevented endothelial IRAK-1 and ICAM-1 expression in db/db mice. Conclusion: These results suggest that miR-146a-5p is involved in the regulation of HG-induced endothelial inflammation via modulation of IRAK-1; indicating that miR-146a-5p may be a novel target for the treatment of diabetic vascular complications.

SUBMITTER: Lo WY

PROVIDER: S-EPMC5539227 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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MicroRNA-146a-5p Mediates High Glucose-Induced Endothelial Inflammation via Targeting Interleukin-1 Receptor-Associated Kinase 1 Expression.

Lo Wan-Yu WY Peng Ching-Tien CT Wang Huang-Joe HJ

Frontiers in physiology 20170802

<b>Background and Aims:</b> Interleukin-1 receptor-associated kinase-1 (IRAK-1) is critical for mediating toll-like receptor and interleukin-1 receptor signaling. In this study, we have examined whether IRAK-1 expression is altered in high glucose (HG)-stimulated human aortic endothelial cells (HAECs), and whether microRNAs (miRs) target IRAK-1 to regulate HG-induced endothelial inflammation. <b>Methods:</b> HAECs were treated with HG for 24 and 48 h. Real-time PCR, Western blot, monocyte adhesi ...[more]

PMID: 28824448

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

Project description:Background and Aims: Increased O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins by O-GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation. Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining. Results: HG upregulated OGT mRNA and protein expression and protein O-GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3'-untranslated region (3'-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O-GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3'-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O-GlcNAcylation levels; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Additionally, siRNA-mediated OGT depletion reduced HG-induced protein O-GlcNAcylation; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion, confirming that HG-induced endothelial inflammation is partially mediated via OGT-induced protein O-GlcNAcylation. These results were validated in vivo: tail-vein injection of miR-200a/200b mimics downregulated endothelial OGT and ICAM-1 expression in db/db mice. Conclusion: miR-200a/200b are involved in modulating HG-induced endothelial inflammation by regulating OGT-mediated protein O-GlcNAcylation, suggesting the therapeutic role of miR-200a/200b on vascular complications in diabetes.

Project description:Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.

Dataset Information

MicroRNA-146a-5p Mediates High Glucose-Induced Endothelial Inflammation via Targeting Interleukin-1 Receptor-Associated Kinase 1 Expression.

Publications

MicroRNA-146a-5p Mediates High Glucose-Induced Endothelial Inflammation via Targeting Interleukin-1 Receptor-Associated Kinase 1 Expression.

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