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.
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.
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.
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.
Article first published online 27 November 2013Supplemental Digital Content is Available in the Text.
Section of Gastroenterology, Department of Medicine, University of Chicago, Chicago, Illinois.
Reprints: John H. Kwon, MD, PhD, University of Chicago, 900 East 57th Street, KCBD 9118, MB#9, Chicago, IL 60637 (e-mail: email@example.com).
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.ibdjournal.org).
Supported by National Institute of Health (NIH; K08 DK078046 to J. H. Kwon; T32 DK07074 to Z. Zhai) and the NIH DDRCC Core Grant (P30 DK42086).
The authors have no conflicts of interest to disclose.
Received July 27, 2013
Accepted October 02, 2013