title: Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium.
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.
  
  
  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.
authors.affiliation: Institute for Medicine and Engineering, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Fang
authors.forename: Peter F
authors.initials: PF
authors.lastname: Davies
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001011:Aorta / Q000276:immunology / Q000378:metabolism; D050197:Atherosclerosis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002478:Cells, Cultured; D018920:Coculture Techniques; D017124:Conserved Sequence; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000276:immunology / Q000378:metabolism*; D017930:Genes, Reporter; D020022:Genetic Predisposition to Disease; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D018836:Inflammation Mediators; C096161:KLF2 protein, human; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha