Abstract
The miR-29 family is involved in fibrosis in multiple organs, including the intestine where miR-29b facilitates TGF-β-mediated up-regulation of collagen in mucosal fibroblasts from Crohn’s disease (CD) patients. Myeloid cell leukemia-1 (MCL-1), a member of the B-cell CLL/Lymphoma 2 (BCL-2) apoptosis family, is involved in liver fibrosis and is targeted by miR-29b via its 3’-UTR in cultured cell lines. We investigate the role of MCL-1 and miR-29b in primary intestinal fibroblasts and tissue from stricturing CD patients. Transfection of CD intestinal fibroblasts with pre-miR-29b resulted in a significant increase in the mRNA expression of MCL-1 isoforms [MCL-1Long (L)/Extra Short (ES) and MCL-1Short (S)], although MCL-1S was expressed at significantly lower levels. Western blotting predominantly detected the anti-apoptotic MCL-1L isoform, and immunofluorescence showed that staining was localised in discrete nuclear foci. Transfection with pre-miR-29b or anti-miR-29b resulted in a significant increase or decrease, respectively, in MCL-1L foci. CD fibroblasts treated with IL-6 and IL-8, inflammatory cytokines upstream of MCL-1, increased the total mass of MCL-1L-positive foci. Furthermore, transfection of intestinal fibroblasts with pre-miR-29b resulted in an increase in mRNA and protein levels of IL-6 and IL-8. Finally, immunohistochemistry showed reduced MCL-1 protein expression in fibrotic CD samples compared to non-stricturing controls. Together, our findings suggest that induction of MCL-1 by IL-6/IL-8 may surmount any direct down-regulation by miR-29b via its 3’-UTR. We propose that an anti-fibrotic miR-29b/IL-6 IL-8/MCL-1L axis may influence intestinal fibrosis in CD. In the future, therapeutic modulation of this pathway might contribute to the management of fibrosis in CD.
Highlights
Crohn’s disease (CD) is characterised by transmural inflammation of the affected bowel, which drives disease progression from an inflammatory to a fibrostenosing phenotype (Rieder et al 2011; Thia et al 2010)
By modulating expression of miR-29b in intestinal fibroblasts isolated from CD patients, we show that MCL-1L expression is altered by this miRNA via the cytokines IL-6 and IL-8 and that MCL-1L levels in stricturing CD tissue samples are lower than in non-stricturing CD samples
To assess the relationship between miR-29b and Myeloid cell leukemia-1 (MCL-1) mRNA expression, intestinal fibroblasts were transfected with NTC or pre-miR-29b and fold change in MCL-1 mRNA was determined relative to the NTC control
Summary
Crohn’s disease (CD) is characterised by transmural inflammation of the affected bowel, which drives disease progression from an inflammatory to a fibrostenosing (stricturing) phenotype (Rieder et al 2011; Thia et al 2010). Intestinal wound healing following acute inflammation-induced damage is a complex sequence of events including inflammatory cell activation of subepithelial fibroblasts. This leads to increased collagen deposition and to a decrease in extracellular matrix (ECM) degradation resulting from an imbalance between tissue-degrading matrix metalloproteinases and their inhibitors (Di Sabatino et al 2009; Graham et al 1988; Regan et al 2000). The production of ECM proteins by activated fibroblasts is critical for intestinal wound healing and the contraction of the wound area (Tomasek et al 2002) Chronic inflammation disturbs this physiological response causing over-production of ECM molecules. The success of such a strategy requires a complete understanding of the anti-apoptotic pathways
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