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miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea

Abstract

Objective Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs.

Design Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells.

Results IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins.

Conclusions Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms.

  • INTESTINAL BARRIER FUNCTION
  • GENE EXPRESSION
  • IRRITABLE BOWEL SYNDROME
  • MOLECULAR BIOLOGY
  • RNA EXPRESSION

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

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Footnotes

  • CM, BKR-J, MV, BN and JS contributed equally.

  • Contributors CM designed the project, performed the research and wrote the paper; BKR-J isolated RNA and protein from all samples and was involved in all research procedures; BL, CA-C, MP, FA and JS were in charge of recruiting patients/controls and collected the biopsies; MLS and SB created T84 stable cell lines and performed overexpression/inhibition analysis and measurements of TEER; BK, MG, WH, RGS and JL were in charge of all statistical analysis regarding RNA sequencing and micro-RNA profiling; RR performed nCounter experiments; IdT was in charge of routinely screening biopsies to exclude signs of inflammation and stained and counted mast cells; GR contributed to essential tools and reagents; BN, MV and JS contributed to the design of the project, supervised all stages of the research and wrote the paper. AMG-s AND ES-R collaborated in the processing of tissue samples and data collection. All authors revised and approved the final version of the manuscript.

  • Funding Supported in part by Fondo de Investigación Sanitaria and CIBERehd, Instituto de Salud Carlos III, Subdirección General de Investigación Sanitaria, Ministerio de Economía y Competitividad: CM08/00229 (BL); CM10/00155 (MP); EII2011-0035 and CD15/00010 (BKR-J); PI12/00314 (CA-C); CP10/00502 and PI13/00935 (MV); PI08/0940, PI11/00716 and PI14/00994 (JS); Ministerio de Educación, Dirección General de Investigación: SAF 2009-07416 (FA); Agència de Gestió d'Ajuts Universitaris i de Recerca, de la Generalitat de Catalunya: 2009 SGR 219 (FA), 2011-BP/A00099 and 2011-BP-A2/00002 (CM); The Rome Foundation Award 2013 (MV); Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas: CB06/04/0021 (FA); Chica and Heinz Schaller Foundation and Deutsche Forschungsgemeinschaft (DFG) in SFB1129 (Project 14) (SB); Brigitte-Schlieben Lange Program from the state of Baden Württemberg, Germany and the Dual Career Support from CellNetworks, Heidelberg, Germany (MS) and the University Hospital Heidelberg (BN, GR). This manuscript results in part from collaboration and network activities promoted under the frame of the international network Genes in IBS Research Network Europe, which has been funded by the COST programme (BM1106, http://www.GENIEUR.eu) and is currently supported by the European Society of Neurogastroenterology and Motility (http://www.ESNM.eu).

  • Competing interests None declared.

  • Ethics approval Vall d'Hebron Ethics Committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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