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Original article
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
  1. Cristina Martínez1,2,3,
  2. Bruno K Rodiño-Janeiro2,3,
  3. Beatriz Lobo2,3,
  4. Megan L Stanifer4,
  5. Bernd Klaus5,
  6. Martin Granzow6,
  7. Ana M González-Castro2,
  8. Eloisa Salvo-Romero2,
  9. Carmen Alonso-Cotoner2,3,7,8,
  10. Marc Pigrau2,3,
  11. Ralph Roeth1,9,
  12. Gudrun Rappold1,
  13. Wolfgang Huber5,
  14. Rosa González-Silos10,
  15. Justo Lorenzo10,
  16. Inés de Torres11,
  17. Fernando Azpiroz2,3,7,8,
  18. Steeve Boulant4,12,
  19. María Vicario2,3,7,8,
  20. Beate Niesler1,8,9,
  21. Javier Santos2,3,7,8
  1. 1Department of Human Molecular Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany
  2. 2Digestive System Research Unit, Institut de Recerca Vall d'Hebron, Barcelona, Spain
  3. 3Facultat de Medicina, Department of Gastroenterology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
  4. 4Schaller Research Group at CellNetworks, Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany
  5. 5European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
  6. 6Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany
  7. 7Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Spain
  8. 8COST Action BM1106 Genes in Irritable Bowel Syndrome (GENIEUR) European Research Network
  9. 9nCounter Core Facility, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany
  10. 10Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany
  11. 11Department of Pathology, Facultat de Medicina, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
  12. 12Research Group ‘Cellular Polarity and Viral Infection’ (F140), German Cancer Research Center (DKFZ), Heidelberg, Germany
  1. Correspondence to Dr Cristina Martinez, Laboratory of Neuro-Immuno-Gastroenterology, Digestive System Research Unit, Department of Gastroenterology, Vall d'Hebron Institut de Recerca, Hospital Universitario Vall d'Hebron, Paseo Vall d'Hebron 119-129, Barcelona 08035, Spain; cristina.martinez{at}med.uni-heidelberg.de, cristina.martinez{at}vhir.org Prof Javier Santos Vicente, Laboratory of Neuro-immuno-gastroenterology, Digestive System Research Unit, Vall d’Hebron Institut de Recerca, Department of Gastroenterology, Hospital Universitario Vall d'Hebron. Paseo Vall d’ Hebron 119-129, Barcelona, Spain; javier.santos{at}vhir.org

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

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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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