Abstract
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by mucosa damage associated with an uncontrolled inflammatory response. This immunological impairment leads to altered inflammatory mediators such as IL-33, which is shown to increase in the mucosa of active UC (aUC) patients. MicroRNAs present a distorted feature in inflamed colonic mucosa and are potential IL-33 regulating candidates in UC. Therefore, we studied the microRNA and mRNA profiles in inflamed colonic samples of UC patients, evaluating the effect of a microRNA (selected by in silico analysis and its expression in UC patients), on IL-33 under inflammatory conditions. We found that inflamed mucosa (n = 8) showed increased expression of 40 microRNAs and 2,120 mRNAs, while 49 microRNAs and 1,734 mRNAs were decreased, as determined by microarrays. In particular, IL-33 mRNA showed a 3.8-fold increase and eight members of a microRNA family (miR-378), which targets IL-33 mRNA in the 3′UTR, were decreased (−3.9 to −3.0 times). We selected three members of the miR-378 family (miR-378a-3p, miR-422a, and miR-378c) according to background information and interaction energy analysis, for further correlation analyses with IL-33 expression through qPCR and ELISA, respectively. We determined that aUC (n = 24) showed high IL-33 levels, and decreased expression of miR-378a-3p and miR-422a compared to inactive UC (n = 10) and controls (n = 6). Moreover, both microRNAs were inversely correlated with IL-33 expression, while miR-378c does not show a significant difference. To evaluate the effect of TNFα on the studied microRNAs, aUC patients with anti-TNF therapy were compared to aUC receiving other treatments. The levels of miR-378a-3p and miR-378c were higher in aUC patients with anti-TNF. Based on these findings, we selected miR-378a-3p to exploring the molecular mechanism involved by in vitro assays, showing that over-expression of miR-378a-3p decreased the levels of an IL-33 target sequence β-gal-reporter gene in HEK293 cells. Stable miR-378a-3p over-expression/inhibition inversely modulated IL-33 content and altered viability of HT-29 cells. Additionally, in an inflammatory context, TNFα decreased miR-378a-3p levels in HT-29 cells enhancing IL-33 expression. Together, our results propose a regulatory mechanism of IL-33 expression exerted by miR-378a-3p in an inflammatory environment, contributing to the understanding of UC pathogenesis.
Highlights
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by chronic and recurrent inflammation of the colon and rectum mucosa
To have an overview of transcripts and microRNAs involved in inflammatory pathways, related to increased IL-33 levels in active UC (aUC) [11], we determined their differential expression in inflamed vs. non-inflamed intestinal mucosa, by microarray assays
To provide an overview of variance in samples given by the inflammatory mucosa context, we performed a Principal Component Analysis (PCA) of microRNAs and mRNAs arrays
Summary
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by chronic and recurrent inflammation of the colon and rectum mucosa. Inactive UC (iUC) correspond to those with normal mucosa, and active UC (aUC) present different degrees of mucosal inflammation classified using the Mayo endoscopic score (MS) [1]. The following elements have been involved in the development of the altered inflammatory response in UC: dysbiosis of gut microbiota [3], the impaired intestinal epithelial barrier function [4] and a loss of tolerance to blood flow agents [5]. These factors lead to the recruitment of inflammatory cells [6] causing persistent tissue damage. Increased IL-33 levels in inflamed intestinal mucosa of aUC patients [11], released mainly from the intestinal colonic epithelium [12], have been proposed as an inflammation disease marker and contributing to disease severity [13]
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