Transgenic Expression of miR-222 Disrupts Intestinal Epithelial Regeneration by Targeting Multiple Genes Including Frizzled-7

Abstract

Defects in intestinal epithelial integrity occur commonly in various pathologies. miR-222 is implicated in many aspects of cellular function and plays an important role in several diseases, but its exact biological function in the intestinal epithelium is underexplored. We generated mice with intestinal epithelial tissue-specific overexpression of miR-222 to investigate the function of miR-222 in intestinal physiology and diseases in vivo. Transgenic expression of miR-222 inhibited mucosal growth and increased susceptibility to apoptosis in the small intestine, thus leading to mucosal atrophy. The miR-222-elevated intestinal epithelium was vulnerable to pathological stress, since local overexpression of miR-222 not only delayed mucosal repair after ischemia/reperfusion-induced injury, but also exacerbated gut barrier dysfunction induced by exposure to cecal ligation and puncture. miR-222 overexpression also decreased expression of the Wnt receptor Frizzled-7 (FZD7), cyclin-dependent kinase 4 and tight junctions in the mucosal tissue. Mechanistically, we identified the Fzd7 messenger ribonucleic acid (mRNA) as a novel target of miR-222 and found that [miR-222/Fzd7 mRNA] association repressed Fzd7 mRNA translation. These results implicate miR-222 as a negative regulator of normal intestinal epithelial regeneration and protection by downregulating expression of multiple genes including the Fzd7. Our findings also suggest a novel role of increased miR-222 in the pathogenesis of mucosal growth inhibition, delayed healing and barrier dysfunction.

Posttranscriptional regulation of intestinal epithelial integrity by noncoding RNAs

“…To determine the in vivo functions of miR‐222 in the intestinal mucosa, a gain‐of‐function transgenic approach was used by generating intestinal epithelial tissue‐specific miR‐222 transgenic (miR222‐Tg) mice. As shown, miR222‐Tg mice exhibit overexpression of miR222/GFP bicistronic RNA specifically in the mucosal tissues of the small intestine, colon, and cecum but it is undetected in stomach mucosa, liver, spleen, kidney, and thymus. There are no significant differences in body weight and gastrointestinal gross morphology in miR222‐Tg mice compared with control littermates.…”

Regulation of Intestinal Epithelial Barrier Function by Long Noncoding RNA uc.173 through Interaction with MicroRNA 29b

“…Biotin-labeled RNA pulldown assays. Biotin-labeled pri-miR-29b was transfected, and 24 h later, whole-cell lysates were collected, mixed with streptavidin-coupled Dynal beads, and incubated on a rotator overnight (52). After the beads were washed thoroughly, the bead-bound RNA was isolated and subjected to RT, followed by Q-PCR analysis.…”

Long Noncoding RNA H19 Impairs the Intestinal Barrier by Suppressing Autophagy and Lowering Paneth and Goblet Cell Function

“…In addition, focal villous necrosis in the small intestinal mucosa was observed 24 hours after CLP, but the damage was repaired within the next 48 hours, as reported previously. 30 Second, we examined changes in the levels of H19 in the small intestinal mucosa from patients with sepsis. Tissue samples from patients without increased gut permeability, mucosal inflammation, or injury/erosions served as controls.…”

“…The immunofluorescence staining procedure was performed according to the method described in our previous publications. 8,30 For experiments using mucosal tissue samples from human beings and mice, dissected and opened intestines were mounted onto a solid surface and fixed in formalin and paraffin. In each tissue sample, more than 5 slides (5-mm thickness per section) were prepared for immunofluorescence staining.…”

“…The epithelial barrier function in vitro was examined by paracellular tracer flux assays using the 12-well Transwell plate (surface area, 1.12 cm 2 ) as described. 30 FITC dextran (70 kilodaltons; Sigma), a membrane-impermeable molecule, served as the paracellular tracer and was added to the apical bathing wells. The basal bathing well had no added tracers and contained the same flux assay medium as in the apical compartment.…”