Abstract
The contribution of miRNA in the pathogenesis of ulcerative colitis (UC) has emerged in the past few decades. Differential miRNA expression has been demonstrated in UC patients, and their ability to target the genes involved in inflammatory pathway has also been explored in recent years. miR-125b and miR-223 have been demonstrated to get upregulated within the colonic mucosa of UC patients. Here, we explored the biological relevance of miR-125b and miR-223 altered expression during UC by identifying the potential gene targets for miR-125b and miR-223. TRAF6 and A20, the signaling molecules involved in the NFκB pathway, were identified as target genes for miR-125b while IKKα was identified as a gene target for miR-223. The colonic mucosal samples from UC patients exhibited a significant rise in miR-125b and miR-223 expression while a subsequent downregulation was observed in the expression of TRAF6, A20, and IKKα. This negative correlation between miRNAs and their respective target genes was validated by co-transfecting miR-125b and miR-223 in HT29 cells. Co-transfection with miR-125b resulted in a marked decline in the expression of TRAF6 and A20, while the miR-223 co-transfected cells exhibited lower IKKα expression levels. Additionally, co-transfection with miR-125b or miR-223 in HT29 cells caused higher p65 and pro-inflammatory cytokines (IL-8 and IL-1β) expression upon LPS stimulation. From our findings, we highlight the possible contribution of miR-125b and miR-223 in regulating the inflammatory response during UC by negatively regulating the expression of TRAF6, A20, and IKKα. Therefore, we conclude that these two miRNAs could be considered as potential candidates for developing promising biomarkers for screening and diagnosis of UC.
Highlights
Ulcerative colitis (UC) is a chronic, idiopathic, and relapsing inflammatory disorder of the gastrointestinal tract and is a major clinical subtype of inflammatory bowel disease (IBD)
TRAF6 was picked up as a target by three prediction tools: TargetScan, DIANAmicroT-CDS, and miRNA.org. It bears an 8mer target site for miR-125b in its 3′UTR at transcript position 1276– 1283 as shown by TargetScan (Figure 2). Both TRAF6 and A20 are key molecules involved in the canonical NFκB pathway and a hyperactive NFκB is reported in the macrophages and epithelial cells derived from inflamed intestinal mucosa [22]
Cytokines Were Highly Upregulated in the Absence of TRAF6 and A20. Both A20 and TRAF6 exhibited a marked decrease in their expression in ulcerative colitis (UC) patients and we have shown that miR-125b efficiently targets the 3′UTR and negatively regulates their expression
Summary
Ulcerative colitis (UC) is a chronic, idiopathic, and relapsing inflammatory disorder of the gastrointestinal tract and is a major clinical subtype of inflammatory bowel disease (IBD). Despite our current understanding regarding the genetic basis of IBD, the monozygotic twin studies have suggested a concordance rate of 10–15% for UC and 30–35% for CD [8] while according to the GWAS, UC accounts only for 16% heritability [9] These reports leave gaps in our apprehension of IBD heritability and highlight the contribution of environmental and epigenetic factors in IBD development and progression. Any alteration in miRNA expression could result in diminished cellular functioning and impaired downstream gene regulation, which implicates the role of miRNA in disease pathogenesis. Distinct miRNA expression has been reported in both the colonic mucosa and peripheral blood of IBD patients, and some of these miRNAs have been implicated to regulate the expression of genes involved in major inflammatory processes [14,15,16]. The biological relevance of miR-125b and miR-223 upregulation and their possible contribution to inflammation during UC was evaluated by looking at the expression of p65 subunit of NF-κB and its downstream pro-inflammatory cytokines in the presence and absence of miR-125b and miR-223 in LPSstimulated HT29 cells
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