Gastroenterology

Gastroenterology

Volume 148, Issue 1, January 2015, Pages 158-169.e8
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
MicroRNA 29 Targets Nuclear Factor-κB–Repressing Factor and Claudin 1 to Increase Intestinal Permeability

https://doi.org/10.1053/j.gastro.2014.09.037Get rights and content

Background & Aims

Some patients with irritable bowel syndrome with diarrhea (IBS-D) have intestinal hyperpermeability, which contributes to their diarrhea and abdominal pain. MicroRNA 29 (MIR29) regulates intestinal permeability in patients with IBS-D. We investigated and searched for targets of MIR29 and investigated the effects of disrupting Mir29 in mice.

Methods

We investigated expression MIR29A and B in intestinal biopsies collected during endoscopy from patients with IBS (n = 183) and without IBS (controls) (n = 36). Levels were correlated with disease phenotype. We also generated and studied Mir29−/− mice, in which expression of Mir29a and b, but not c, is lost. Colitis was induced by administration of 2,4,6-trinitrobenzenesulfonic acid; intestinal tissues were collected and permeability was assessed. Microarray analysis was performed using tissues from Mir29−/− mice. Changes in levels of target genes were measured in human colonic epithelial cells and small intestinal epithelial cells after knockdown of MIR29 with anti-MIRs.

Results

Intestinal tissues from patients with IBS-D (but not IBS with constipation or controls) had increased levels of MIR29A and B, but reduced levels of Claudin-1 (CLDN1) and nuclear factor-κB–repressing factor (NKRF). Induction of colitis and water avoidance stress increased levels of Mir29a and Mir29b and intestinal permeability in wild-type mice; these increased intestinal permeability in colons of far fewer Mir29−/− mice. In microarray and knockdown experiments, MIR29A and B were found to reduce levels of NKRF and CLDN1 messenger RNA, and alter levels of other messenger RNAs that regulate intestinal permeability.

Conclusions

Based on experiments in knockout mice and analyses of intestinal tissue samples from patients with IBS-D, MIR29 targets and reduces expression of CLDN1 and NKRF to increase intestinal permeability. Strategies to block MIR29 might be developed to restore intestinal permeability in patients with IBS-D.

Section snippets

miR-29a/b-/- Mice

To determine the essential role of miR-29 in vivo, we generated miR-29a/b knockout mice. Homozygous floxed miR-29ab1 mice (C57BL/6 strain) were generated by 2 homologous recombination arms that were amplified by polymerase chain reaction on 129 SvJ/X1 genomic DNA, a 5′ sequence of 4171 bp, and a 3′ sequence of 3857 bp. The genomic fragment to be deleted, which had 600 bp, and which contained the miR-29a and miR-29b1 regions, was amplified and cloned in between 2 loxP sites, in a pFlox vector (

Additional Methods

Detailed methodology is described in the Supplementary Methods.

Intestinal Hyperpermeability and miR-29 Expression in Humans

We enrolled 233 subjects; 219 (94%) of which completed the study; 14 dropped out, including 12 IBS patients and 2 controls. Of the 219, there were 109 IBS-D patients (28.6 ± 2.9 years old, 34 male and 75 female); 74 constipation-predominant IBS (IBS-C) patients (30.4 ± 4.3 years, 20 male and 54 female); and 36 healthy controls (mean age 31.5 ± 3.6 years; 10 male and 26 female). There was no significant difference in age or sex between the groups (controls, IBS).

Increased intestinal permeability

Facilitation of Intestinal Barrier Integrity in miRNA-29a/b−/− Mice Via Claudin-1 and Nuclear Factor-κB–Repressing Factor Signaling

To confirm that CLDN1 and NKRF are target gene regulators of intestinal permeability in miR-29a/b−/− mice, we performed in situ hybridization on colon tissue sections from WT mice and miR-29a/b−/− mice labeled with a probe specific for mature miR-29a and miR-29b and its co-localization with CLDN1 (Figure 4A and B). The miR-29a expression was lower in miR-29a/b−/− mice (Figure 4Ba) than in controls (Figure 4Aa). There was an increase in CLDN1 expression in miR-29a/b−/− mouse colon (Figure 4Bb)

Discussion

The novel findings of our current study show that silencing miR-29 family in knockout mice (miR-29a/b-/-) restores intestinal permeability and the associated pathologic hallmarks of increased intestinal permeability. To our knowledge, our findings are the first translational datasets (in vitro, in vivo, and human ex vivo) to demonstrate that suppression of a cluster miRNA can reverse increased intestinal permeability; to identify miR-29 targets in miR-29a/b-/- mice and the function of 1238

References (40)

  • T. Inai et al.

    Claudin-1 contributes to the epithelial barrier function in MDCK cells

    Eur J Cell Biol

    (1999)
  • R. Sen et al.

    Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism

    Cell

    (1986)
  • G. Bamias et al.

    New insights into the dichotomous role of innate cytokines gut homeostasis and inflammation

    Cytokine

    (2012)
  • P.M. Majewski et al.

    Cooperative role of NF-{kappa}B and poly(ADP-ribose) polymerase 1 (PARP-1) in the TNF-induced inhibition of PHEX expression in osteoblasts

    J Biol Chem

    (2010)
  • E.A. Mayer

    Irritable bowel syndrome

    N Engl J Med

    (2008)
  • M. Camilleri et al.

    Intestinal barrier function in health and gastrointestinal disease

    Neurogastroenterol Motil

    (2012)
  • Q. Zhou et al.

    New insights into visceral hypersensitivity-clinical implications in IBS

    Nat Rev Gastroenterol Hepatol

    (2011)
  • R.C. Spiller et al.

    Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome

    Gut

    (2000)
  • S.P. Dunlap et al.

    Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndrome

    Am J Gastroenterol

    (2007)
  • Q. Zhou et al.

    MicroRNA-29a regulates intestinal membrane permeability in patients with irritable bowel syndrome

    Gut

    (2010)
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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by grants from the National Institutes of Health from National Institute of Diabetes and Digestive and Kidney Diseases (DK099052); National Center for Complementary and Alternative Medicine (AT005291); and from the Department of Veteran Affairs.

    Author names in bold designate shared co-first authorship.

    Authors share co-first authorship.

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