Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium

Arterioscler Thromb Vasc Biol. 2012 Apr;32(4):979-87. doi: 10.1161/ATVBAHA.111.244053. Epub 2012 Jan 19.

Abstract

Objective: Endothelial transcription factors Krüppel-like factor 4 (KLF4) and KLF2 are implicated in protection against atherogenesis. Steady-state microRNA (miR) regulation of KLFs in vivo is accessible by screening region-specific endothelial miRs and their targets.

Methods and results: A subset of differentially expressed endothelial miRs was identified in atherosusceptible versus protected regions of normal swine aorta. In silico analyses predicted highly conserved binding sites in the 3'-untranslated region (3'UTR) of KLF4 for 5 miRs of the subset (miR-26a, -26b, -29a, -92a, and -103) and a single binding site for a miR-92a complex in the 3'UTR of KLF2. Of these, only miR-92a knockdown and knock-in resulted in responses of KLF4 and KLF2 expression in human arterial endothelial cells. Dual luciferase reporter assays demonstrated functional interactions of miR-92a with full-length 3'UTR sequences of both KLFs and with the specific binding elements therein. Two evolutionarily conserved miR-92a sites in KLF4 3'UTR and 1 site in KLF2 3'UTR were functionally validated. Knockdown of miR-92a in vitro resulted in partial rescue from cytokine-induced proinflammatory marker expression (monocyte chemotactic protein 1, vascular cell adhesion molecule-1, E-selectin, and endothelial nitric oxide synthase) that was attributable to enhanced KLF4 expression. Leukocyte-human arterial endothelial cell adhesion experiments supported this conclusion. In swine aortic arch endothelium, a site of atherosusceptibility where miR-92a expression was elevated, both KLFs were expressed at low levels relative to protected thoracic aorta.

Conclusions: miR-92a coregulates KLF4 and KLF2 expression in arterial endothelium and contributes to phenotype heterogeneity associated with regional atherosusceptibility and protection in vivo.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Aorta / immunology
  • Aorta / metabolism
  • Atherosclerosis / genetics*
  • Atherosclerosis / immunology
  • Atherosclerosis / metabolism
  • Atherosclerosis / prevention & control
  • Base Sequence
  • Binding Sites
  • Cell Adhesion
  • Cells, Cultured
  • Coculture Techniques
  • Conserved Sequence
  • Endothelial Cells / immunology
  • Endothelial Cells / metabolism*
  • Endothelium, Vascular / immunology
  • Endothelium, Vascular / metabolism*
  • Genes, Reporter
  • Genetic Predisposition to Disease
  • Humans
  • Inflammation Mediators / metabolism
  • Kruppel-Like Transcription Factors / genetics*
  • Kruppel-Like Transcription Factors / metabolism
  • Leukocytes / immunology
  • Leukocytes / metabolism
  • MicroRNAs / metabolism*
  • Phenotype
  • RNA Interference
  • RNA, Messenger / metabolism
  • Swine
  • Transfection
  • Tumor Necrosis Factor-alpha / metabolism
  • Up-Regulation

Substances

  • 3' Untranslated Regions
  • GKLF protein
  • Inflammation Mediators
  • KLF2 protein, human
  • Kruppel-Like Transcription Factors
  • MIRN92 microRNA, human
  • MicroRNAs
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha