Effect of Alcohol on miR‐212 Expression in Intestinal Epithelial Cells and Its Potential Role in Alcoholic Liver Disease

Abstract

Background and Aims: Alcohol-induced gut leakiness is a key factor in alcoholic liver disease (ALD); it allows endotoxin to enter the circulation and initiate liver damage. Zonula occludens 1 (ZO-1) protein is a major component of tight junctions that regulates intestinal permeability. microRNAs (miRNAs) are recently discovered regulatory molecules that inhibit expression of their target genes. The aims of our study were: (i) to investigate the effect of alcohol on miRNA-212 (miR-212) and on expression of its predicted target gene, ZO-1, (ii) to study the potential role of miR-212 in the pathophysiology of ALD in man.Methods: Using a TaqMan miRNA assay system, we measured miR-212 expression levels in colon biopsy samples from patients with ALD and in Caco-2 cells (a human intestinal epithelial cell line) treated with or without EtOH. We measured ZO-1 protein levels using western blots. ZO-1 mRNA was assayed using real-time PCR. Intestinal barrier integrity was measured using fluorescein sulfonic acid clearance and immunofluorescent staining for ZO-1.Results: Ethanol increased miR-212 expression, decreased ZO-1 protein levels, disrupted tight junctions, and increased the permeability of monolayers of Caco-2 cells. An miR-212 over-expression is correlated with hyperpermeability of the monolayer barrier. miR-212 levels were higher in colon biopsy samples in patients with ALD than in healthy controls; ZO-1 protein levels were lower.Conclusion: These data suggest a novel mechanism for alcohol-induced gut leakiness, one in which EtOH induces miR-212 over-expression which causes gut leakiness by down-regulating ZO-1 translation. This mechanism is a potential therapeutic target for leaky gut in patients with or at risk for ALD.

A sideways glance. Alcoholic breakdown of barriers: how ethanol can initiate a landslide towards disease

“…It thus appears that ethanol-induced miR-212 expression mediates inhibition of ZO-1 translation, although not affecting transcription, a mechanism already proposed for other miRNAs [14]. To substantiate these findings the authors found that sigmoid colon biopsies of ALD patients that exhibit increased intestinal permeability, also expressed higher levels of miR-212 and lower amounts of ZO-1 protein compared to healthy controls [20]. This is an interesting novel mechanism of TJ protein regulation by microRNAs that awaits further investigation to elucidate its role in normal physiological conditions.…”

“…[17] A novel mechanism of regulation of proteins involved in TJ maintenance has been proposed to involve microRNAs. It was recently shown that treatment of human intestinal Caco-2 cells with ethanol under conditions that increase TJs permeability, led to over-expression of the microRNA miR-212 and to decreased expression of ZO-1 protein levels, while the mRNA levels were unaffected [20]. It thus appears that ethanol-induced miR-212 expression mediates inhibition of ZO-1 translation, although not affecting transcription, a mechanism already proposed for other miRNAs [14].…”

MicroRNAs in Liver Disease: Bench to Bedside

“…[113][114][115][116][117] Ethanol has been shown to repress miRs 9, 21, 153 and 335 in neural stem cells and neural progenitor cells thus altering cellular development and maturation. 113 Alcohol has been found to increase miR-212 expression and its over-expression is correlated with hyperpermeability of monolayer barrier (tight junction) by down-regulating protein Zonula occludens 1 translation.…”

“…113 Alcohol has been found to increase miR-212 expression and its over-expression is correlated with hyperpermeability of monolayer barrier (tight junction) by down-regulating protein Zonula occludens 1 translation. 114 Long-term exposure to alcohol can also induce endothelin 1 (ET-1) and hypoxia-inducible factor 1 and inhibit expression of miR-199 in liver sinusoidal endothelial cells. 115 MiR-199 is involved in ET-1 expression in human endothelial cells and functions as a negative regulator of ET-1 transcription and thus vascular tone in ALD.…”

Cell-secreted vesicles containing microRNAs as regulators of gamete maturation

“…Based on results by Schauer and coworkers, these miRNAs are altered by LH induction in follicular fluid as well as in granulosa cells and are involved in cell survival and final differentiation. Predicted targets include PTEN and BMPR2 (miR-21; Qin et al 2009, Schauer et al 2013, involved in regulating granulosa cell proliferation (Fan et al 2008) and maturation of the cumulus-oocyte-complex (Assou et al 2006) and TJP1 (miR-212; Tang et al 2008) involved in cell-cell interactions and regulation of tight junctions in ovarian surface epithelial cells (Zhu et al 2004).…”