Integrated miRNA and mRNA expression profiling in inflamed colon of patients with ulcerative colitis.

Jan Van Der Goten,Paul Rutgeerts,Kathleen Machiels,Willem-Jan Wollants,Gert De Hertogh,Marc Ferrante,Leentje Van Lommel,Frans Schuit,Séverine Vermeire,Vicky De Preter,Katleen Lemaire,Ingrid Arijs,Gert Van Assche,Wiebe Vanhove,Mathias Chamaillard

doi:10.1371/journal.pone.0116117

Abstract

BackgroundUlcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.MethodologyColonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.ResultsWhen comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.ConclusionDifferential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.

Highlights

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) and is characterized by a diffuse mucosal inflammation extending proximally from the rectum to a varying degree in the colon [1]
Cluster I included all the controls, all 7 inactive UC patients and only 1/10 active UC patient, and cluster II contained 9/10 active UC patients. These findings indicate distinctive miRNA expression profiles based on colonic inflammatory load
If we compared the miRNA expression profiles of all UC patients vs. normal controls, four mature miRNAs were significantly differentially expressed: hsamiR-675-5p was significantly upregulated in UC vs. controls, while hsa-miR-378a5p, hsa-miR-196b-5p and hsa-miR-10b-5p were significantly downregulated

Summary

Introduction

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) and is characterized by a diffuse mucosal inflammation extending proximally from the rectum to a varying degree in the colon [1]. It is widely accepted that IBD is the result of an inadequate and ongoing activation of the mucosal immune system to the luminal microbiota in genetically predisposed subjects This complex interaction of genetic, immune, and environmental factors causing IBD is reflected in broad gene expression changes which can distinguish IBD from controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. We identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs controls and found a highly significant inverse correlation between hsa-miR-200c-3p and PLOS ONE | DOI:10.1371/journal.pone.0116117. We identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and PLOS ONE | DOI:10.1371/journal.pone.0116117 December 29, 2014

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