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IBS is a functional GI disorder characterised by abdominal bloating, increased visceral pain perception and an altered defaecation pattern in the absence of an organic cause. The underlying pathophysiological mechanisms that lead to abdominal pain and chronic symptoms in IBS are not fully understood. A common finding is the presence of increased mucosal intestinal permeability and an altered distribution of cell-to-cell adhesion proteins in the intestinal epithelium.1 In particular, patients with diarrhoea-predominant IBS (IBS-D)2 suffer from increased permeability, and this increase correlates with aberrant visceral sensitivity.3 Based on these observations, it is currently hypothesised that impairment of the intestinal barrier integrity facilitates the entrance of food or microbial antigens into the intestinal mucosa, thereby stimulating the mucosal immune system. Accordingly, subsets of patients with IBS show higher numbers and an increased activation of mucosal immunocytes, particularly mast cells. Bioactive mediators released by these cells, including proteases, histamine and prostanoids, participate in the perpetuation of the permeability dysfunction4 and contribute to the activation and sensitisation of (sensory) neurons5 leading to aberrant abdominal pain perception and changes in bowel habits.
Both endogenous (genetic) and environmental factors can lead to intestinal barrier impairment, but psychological stress and infectious gastroenteritis are probably the best known and strongest triggers. Increased colonic paracellular permeability has indeed been extensively observed in both chronic and acute stress murine models,6 and the connection between stress and permeability is slowly evolving in humans.7 On the other hand, infectious gastroenteritis is a common trigger for IBS, as the risk of developing IBS after acute gastroenteritis is sixfold greater than that for an individual with no prior GI infection and occurs in ∼10% of individuals post infection.8 It is still not clear why an acute enteric infection can lead to long-term consequences such as hyperpermeability and IBS symptoms in predisposed individuals. In fact, insight in the mechanisms that modulate mucosal permeability at molecular level by altering the expression of cell-to-cell adhesion proteins is largely lacking.
A lot of attention has recently been drawn to microRNAs (miRNAs). These small regulatory non-coding RNA molecules of typically 22 nucleotides are encoded by the genome and inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs (mRNAs) or otherwise inhibiting their translation into proteins. miRNAs generally recognise their target mRNAs via a limited region on the three prime untranslated region (3′-UTR)-called the seed sequence.9 Through seed pairing, miRNAs can generally target hundreds of mRNAs. It is now estimated that the human genome may encode more than 1000 miRNAs that can regulate the expression of up to 60% of our genes.9 The identification of true miRNA targets has been the topic of extensive research, and there is emerging evidence that the full impact of some miRNAs upon their targets may only be seen in very specific disease conditions or in response to certain environmental stimuli.
The most successful study on the role of miRNAs in IBS was carried out by Zhou et al10 who reported modulation of intestinal permeability by miR-29a via downregulation of glutamine synthetase. Glutamine plays a crucial role in the maintenance of intestinal barrier function in animals and humans,11 and its depletion leads to villus atrophy, decreased expression of tight junction proteins and increased intestinal permeability. Ex vivo, glutamine administration increased the expression of tight junction protein claudin-1 in the colonic mucosa of patients with IBS-D,12 and further studies are required to evaluate glutamine supplementation in clinical practice. Besides miR-29a, others found evidence for miRNAs modulating serotonin-related genes,13 ,14 the nociceptor TRPV115 or inflammation16 in IBS.
In Gut, Martínez et al17 convincingly identified both transcriptional and post-transcriptional mechanisms that contribute to barrier dysfunction in the jejunum of patients with IBS-D. The authors applied a comprehensive approach including the identification of both mRNA and miRNA expression patterns in jejunal biopsies of patients with IBS-D versus healthy individuals, followed by a targeted overexpression or downregulation of the newly identified miRNAs in epithelial cell lines yielding crucial novel insight into their functional role. First, at mRNA level, several canonical signalling pathways were associated with IBS-D including genes involved in barrier integrity such as tight junction signalling, adherens junction signalling and actin cytoskeleton signalling. Second, comparison of IBS-D and control jejunal biopsies revealed downregulation of miR-125b and miR-16 in IBS-D, while their respective predicted target proteins, cingulin and claudin 2, were upregulated. Overexpression or inhibition of miR-125b and miR-16 in human epithelial cells confirmed their repressive effects on cingulin and claudin 2 protein expression, respectively. Finally, downregulation of miR-125b and miR-16 impaired epithelial barrier integrity of human intestinal epithelial cells, and this effect was due to the disorganisation of tight junction proteins. It is noteworthy that the authors confirmed their RNAseq and miRNA results by various techniques, including qPCR and the nCounter and Agilent miRNA microarray platform, making their data more robust. Despite these efforts, the authors were not able to confirm previously reported findings.10 ,14 ,15 Indeed, the major drawback of miRNA results in IBS reported so far is the variability in reproducibility, most likely due to small sample sizes, different regions in the gut that were studied, or due to different methodologies. In the manuscript by Martínez et al, jejunal biopsies of 26 healthy individuals and 43 patients with IBS were investigated, while epigenetic studies in immune or cancer-related diseases typically analyse hundreds of patients and controls. Moreover, these authors studied miRNA expression in the jejunum while the majority of studies in IBS are performed on colon tissue. It is still unclear whether IBS is a disorder of the small or the large intestine or both and if experimental results obtained in the small intestine reflect what is happening in the large intestine and vice versa. Of note, even though the results of Martínez et al are of extreme interest to gain mechanistic insight in the molecular mechanisms underlying barrier dysfunction in IBS, it should be noted that these miRNAs are not gene-specific and also target other—yet unexplored––biological pathways. Moreover, genes contain various miRNA-binding sites, and the levels of gene expression are regulated by a combination of miRNAs and transcription factors that add to the complexity. Finally, it would be of great interest to explore if the dysregulation of miRNAs in the intestinal cells is also reflected in cell-derived extracellular vesicles such as exosomes. Exosomes, 40–100 nm nanosized vesicles that are released from practically all cell types into blood or urine, have been shown to act as mediators for cell-to-cell communication because of the regulatory functions of their content. As high levels of exosomes are found in several body fluids, they may serve as a potential source of non-invasive biomarkers for hyperpermeability or other pathophysiological mechanisms underlying IBS.
miRNA-based therapies for IBS?
miRNAs targeting genes involved in hyperpermeability or neuronal hypersensitivity provide a new and potential powerful candidate for therapeutic intervention in IBS. But, even though the information about miRNA biology has significantly enriched over the years, we still do not completely understand the mechanism of miRNA gene regulation. Various groups across the world and pharmaceutical companies are conducting research to explore miRNA-based therapies and improve delivery vehicles. Consequently, some miRNAs have already entered the preclinical and clinical stage, and the very first clinical trial results of a miRNA replacement therapy in patients with thoracic cancer18 are soon expected.
Altogether, miRNA expression profiles are altered in patients with IBS-D, leading to increased mucosal permeability. Further study on the identification and functional role of miRNAs in large IBS cohorts will lead to a better understanding of the underlying pathophysiological mechanisms. This novel insight may yield the identification of a panel of biomarkers that allows stratification of IBS subgroups. In the future, specific inhibitors of miRNA or miRNA analogues that target gene expression may potentially become available to treat IBS.
Funding This work was funded by a FWO research grant G0A9516N.
Competing interests None declared.
Provenance and peer review Commissioned; internally peer reviewed.
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