title: NOD2 expression is regulated by microRNAs in colonic epithelial HCT116 cells.
abstract: BACKGROUND
  Crohn's disease (CD) is associated with defective sensing of pathogens in genetically susceptible individuals. Nucleotide-binding oligomerization domain containing 2 (NOD2) mutations in coding regions are strongly linked to CD pathogenesis. Our laboratory has reported that microRNAs (miRNAs) are differentially expressed in CD. However, miRNA regulation of NOD2 remains unknown. This study was designed to determine whether miRNAs regulate NOD2 expression as well as downstream nuclear factor kappaB activation and inflammatory responses in colonic epithelial HCT116 cells.
  
  
  METHODS
  NOD2 and miRNA expression in stimulated HCT116 cells were assessed by quantitative reverse transcription-polymerase chain reaction. Regulation of NOD2 expression by miRNAs was determined by luciferase reporter construct assays and transfection of specific miRNA mimics. Regulation of NOD2 signaling and immune response by miRNAs was assessed by transfection of mimics followed by muramyl dipeptide stimulation.
  
  
  RESULTS
  Muramyl dipeptide-induced increases in NOD2, interleukin-8, and CXCL3 expression were inversely associated with miRNA expression. Overexpression of miR-192, miR-495, miR-512, and miR-671 suppressed NOD2 expression, muramyl dipeptide-mediated NF-κB activation, and messenger RNA expressions of interleukin-8 and CXCL3 in HCT116 cells. A single-nucleotide polymorphism (rs3135500) located in the NOD2 3'-untranslated region significantly reduced miR-192 effects on NOD2 gene expression.
  
  
  CONCLUSIONS
  To our knowledge, this is the first report demonstrating that miRNAs regulate NOD2 and its signaling pathway. Four miRNAs downregulate NOD2 expression, suppress NF-κB activity, and inhibit interleukin-8 and CXCL3 messenger RNA expression. Treatment of CD with miRNAs may represent a potential anti-inflammatory therapeutic strategy in CD patients with and without NOD2 gene mutations.
authors.affiliation: Section of Gastroenterology, Department of Medicine, University of Chicago, Chicago, Illinois.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Chuang
authors.forename: Jim C
authors.initials: JC
authors.lastname: Chuang
authors.forename: Zili
authors.initials: Z
authors.lastname: Zhai
authors.forename: Feng
authors.initials: F
authors.lastname: Wu
authors.forename: John H
authors.initials: JH
authors.lastname: Kwon
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D020413:3' Untranslated Regions; D016209:Interleukin-8; C000595430:MIRN512 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger; D008156:Luciferases