Nijhuis Anke, Curciarello Renata, Mehta Shameer, Feakins Roger, Bishop Cleo L, Lindsay James O, Silver Andrew
Centre for Genomics and Child Health and National Centre for Bowel Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark St, Whitechapel, E1 2AT, London, UK.
Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark St, Whitechapel, E1 2AT, London, UK.
Cell Tissue Res. 2017 May;368(2):325-335. doi: 10.1007/s00441-017-2576-1. Epub 2017 Feb 11.
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.
miR-29家族参与多个器官的纤维化过程，包括肠道，在肠道中miR-29b促进转化生长因子-β（TGF-β）介导的克罗恩病（CD）患者黏膜成纤维细胞中胶原蛋白的上调。髓样细胞白血病-1（MCL-1）是B细胞慢性淋巴细胞白血病/淋巴瘤2（BCL-2）凋亡家族的成员，参与肝纤维化，在培养细胞系中通过其3'-非翻译区（3'-UTR）被miR-29b靶向作用。我们研究了MCL-1和miR-29b在狭窄性CD患者的原代肠道成纤维细胞和组织中的作用。用pre-miR-29b转染CD肠道成纤维细胞导致MCL-1亚型[MCL-1长（L）/超短（ES）和MCL-1短（S）]的mRNA表达显著增加，尽管MCL-1S的表达水平显著较低。蛋白质免疫印迹法主要检测到抗凋亡的MCL-1L亚型，免疫荧光显示染色定位于离散的核灶。用pre-miR-29b或抗-miR-29b转染分别导致MCL-1L灶显著增加或减少。用MCL-1上游的炎性细胞因子白细胞介素-6（IL-6）和白细胞介素-8（IL-8）处理CD成纤维细胞，增加了MCL-1L阳性灶的总量。此外，用pre-miR-29b转染肠道成纤维细胞导致IL-6和IL-8的mRNA和蛋白质水平增加。最后，免疫组织化学显示，与非狭窄对照组相比，纤维化CD样本中MCL-1蛋白表达降低。总之，我们的研究结果表明，IL-6/IL-8诱导MCL-1可能克服miR-29b通过其3'-UTR的任何直接下调作用。我们提出，抗纤维化的miR-29b/IL-6/IL-8/MCL-1L轴可能影响CD中的肠道纤维化。未来，对该途径的治疗性调节可能有助于CD纤维化的管理。
