Inhibition of miR-378a-3p by Inflammation Enhances IL-33 Levels: A Novel Mechanism of Alarmin Modulation in Ulcerative Colitis

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.

NGF/TRKA Decrease miR-145-5p Levels in Epithelial Ovarian Cancer Cells

“…For miR over-expression assays we used the pGPGLenti-human cytomegalovirus-green fluorescent protein (CMV-GFP)-puro vector, previously validated for microRNA expression [ 83 ]. After amplification of the primiR-145 sequence (300 pb flanking the miR-145 stemloop: MI0000461), this insert was cloned in the SalI site in forward sense (pGPG-145).…”

emiRIT: a text-mining-based resource for microRNA information

“…To help users navigate the miRNA information in the literature, emiRIT just captures terms about cellular processes and verifies if the terms correspond to any GO term using exact string matching. For example, the UCL annotations for miR-378a-3p included involvement in the negative regulation of cytokine production involved in inflammatory response (GO:1900016) with evidence from PMID:31824476 ( 70 ). From this abstract, emiRIT extracts the mention of IL-33 being a target of miR-378.…”

Integrative Analysis of CircRNA Expression and Associated ceRNA Network in Graves’ Disease

“…The miR-378 family has not been reported in studies of GD patients, but was reported by Shi Haizhong et al [18]. It is suggested that the low expression of the miR-378 family enhanced the levels of its downstream molecule IL-33 and played a role in the proin ammatory response of the autoimmune disease ulcerative colitis (UC).…”