Original ResearchFull Report: Basic and Translational—Alimentary TractMicroRNA 29 Targets Nuclear Factor-κB–Repressing Factor and Claudin 1 to Increase Intestinal Permeability
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
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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.
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Authors share co-first authorship.