bwge2017

Authors

S. VAN REMOORTEL (1), R. ARORA (2), H. DE SCHEPPER (3), J. TIMMERMANS (2), R. BUCKINX (2) / [1] University of Antwerp, Antwerpen, Belgium, Veterinary Sciences, [2] University of Antwerp, Antwerp, Belgium, Veterinary Sciences, [3] Antwerp University Hospital, Edegem, Belgium, Gastroenterology and Hepatology

Introduction
G protein-coupled receptors (GPCR’s) are leading targets in drug discovery because of their extensive regulatory functions and profound molecular diversity. In this respect, Mas-related G protein-coupled receptors (MRGPR) are a promising family of GPCR’s that are involved in peripheral pain perception and mast cell physiology. Our previous results show that the expression of multiple murine MRGPR members in enteric neurons and mucosal mast cells markedly changes during intestinal inflammation. This suggests a role in GI neuro-immune communication, which is involved in both pathological and functional gastro-intestinal (GI) disorders (Avula et al. 2011; 2013).

Aim

Because nothing is known about MRGPR expression in the human GI tract, our aim was to characterize their expression in mucosal biopsies and evaluate expressional changes in the GI tract of patients with irritable bowel syndrome (IBS).

Methods
Mucosal biopsies from the terminal ileum were collected during colonoscopy and stored in the RNA stabilizer RNAlater© until further processing. Individuals were categorized as healthy control (n=10) or IBS (n= 18) according to the Rome III criteria. We selected and evaluated a panel of five genes as candidate reference genes, in compliance with MIQE qPCR quality standards (Bustin et al., 2009), and then determined the expression for MRGPRD, MRGPRE, MRGPRF, MRGPRX1-4 and MAS1L.

Results
GeNorm analysis (Vandesompele et al. 2002) showed that reference genes HPRT1 and PPIA, with a combined M-value of 0.255, are expressed most stable in the mucosal biopsies. Among the target genes, MRGPRD, MRGPRE, MRGPRF, MRGPRX2 and MAS1L are clearly expressed in the GI mucosa, while the other target genes are not expressed. Moreover, MRGPRD and MRGPRE expression showed a trend towards downregulation in IBS individuals compared to healthy controls, with less variability between patient samples. This trend is mainly attributed by a reduced expression of MRGPRD and MRGPRE in the IBS-C subtype.

Conclusions
From a selected panel of candidate reference genes, we show that HPRT1 and PPIA are most suitable to use as reference genes for expression normalization in mucosal biopsies from the terminal ileum. We provide first evidence for the expression of multiple MRGPR members in the human ileum, which warrants further work on the (sub)cellular localization of these receptors. Moreover, the plasticity of MRGPR expression suggests a possible role for these receptors in GI (patho)physiology.