Journal Article

MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro

Shi, Congzhu, Civelek, Mete, Catravas, John, Manduchi, Elisabetta, Davies, Peter F., Fang, Yun

2010

Overview

A chronic proinflammatory state precedes pathological change in arterial endothelial cells located within regions of susceptibility to atherosclerosis. The potential contributions of regulatory microRNAs to this disequilibrium were investigated by artery site-specific profiling in normal adult swine. Expression of endothelial microRNA10a (miR-10a) was lower in the athero-susceptible regions of the inner aortic arch and aorto-renal branches than elsewhere. Expression of Homeobox A1 (HOXA1), a known miR-10a target, was up-regulated in the same locations. Endothelial transcriptome microarray analysis of miR-10a knockdown in cultured human aortic endothelial cells (HAEC) identified IκB/NF-κB–mediated inflammation as the top category of up-regulated biological processes. Phosphorylation of IκBα, a prerequisite for IκBα proteolysis and NF-κB activation, was significantly up-regulated in miR-10a knockdown HAEC and was accompanied by increased nuclear expression of NF-κB p65. The inflammatory biomarkers monocyte chemotactic protein 1 (MCP-1), IL-6, IL-8, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were elevated following miR-10a knockdown. Conversely, knockin of miR-10a (a conservative 25-fold increase) inhibited the basal expression of VCAM-1 and E-selectin in HAEC. Two key regulators of IκBα degradation—mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and β-transducin repeat-containing gene (βTRC)—contain a highly conserved miR-10a binding site in the 3' UTR. Both molecules were up-regulated by miR-10a knockdown and suppressed by miR-10a knockin, and evidence of direct miR-10a binding to the 3' UTR was demonstrated by luciferase assay. Comparative expression studies of endothelium located in athero-susceptible aortic arch and athero-protected descending thoracic aorta identified significantly up-regulated MAP3K7, βTRC, phopho-IκBα, and nuclear p65 expression suggesting that the differential expression of miR-10a contributes to the regulation of proinflammatory endothelial phenotypes in athero-susceptible regions in vivo.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: 3' untranslated regions / adults / Animals / aorta / Aorta, Thoracic - metabolism / Aorta, Thoracic - pathology / Atherosclerosis / Atherosclerosis - genetics / beta-Transducin Repeat-Containing Proteins - genetics / beta-Transducin Repeat-Containing Proteins - metabolism / Binding sites / Biological Sciences / Biomarkers / Blotting, Western / cell adhesion / Cell Line / Cells / Cells, Cultured / chemokine CCL2 / Endothelial cells / Endothelial Cells - metabolism / Endothelial Cells - pathology / Endothelium / Endothelium - metabolism / Endothelium - pathology / Gene Expression Profiling / Gene expression regulation / Gene Knockdown Techniques / Genes / Genetic Predisposition to Disease / Genotype & phenotype / Hogs / Homeodomain Proteins - genetics / Homeodomain Proteins - metabolism / Humans / I-kappa B Proteins - metabolism / IKappaB kinase / In Situ Hybridization, Fluorescence / inflammation / Inflammation - genetics / interleukin-6 / interleukin-8 / Kinases / luciferase / MAP Kinase Kinase Kinases - genetics / MAP Kinase Kinase Kinases - metabolism / microarray technology / MicroRNA / MicroRNAs - genetics / MicroRNAs - metabolism / Molecules / NF-kappa B - metabolism / NF-KappaB Inhibitor alpha / Oligonucleotide Array Sequence Analysis / Phenotype / Phenotypes / phosphorylation / proteolysis / Reverse Transcriptase Polymerase Chain Reaction / Ribonucleic acid / RNA / Swine / transcription factor NF-kappa B / Transcription Factors - genetics / Transcription Factors - metabolism / transcriptome / Untranslated regions / Up regulation