MiR-144 Increases Intestinal Permeability in IBS-D Rats by Targeting OCLN and ZO1

Abstract

Background/Aims: Irritable bowel syndrome with diarrhoea (IBS-D) is a chronic, functional bowel disorder characterized by abdominal pain or diarrhoea and altered bowel habits, which correlate with intestinal hyperpermeability. MicroRNAs (miRNAs) are involved in regulating intestinal permeability in IBS-D. However, the role of miRNAs in regulating intestinal permeability and protecting the epithelial barrier remains unclear. Our goals were to (i) identify differential expression of miRNAs and their targets in the distal colon of IBS-D rats; (ii) verify in vitro whether occludin (OCLN) and zonula occludens 1 (ZO1/TJP1) were direct targets of miR-144 and were down-regulated in IBS-D rats; and (iii) determine whether down-regulation of miR-144 in vitro could reverse the pathological hallmarks of intestinal hyperpermeability via targeting OCLN and ZO1. Methods: The IBS-D rat model was established using 4% acetic acid and evaluated by haematoxylin-eosin (HE) staining. The distal colon was obtained in order to perform miRNA microarray analysis and to isolate and culture colonic epithelial cells. When differential expression of miRNA was found, the results were verified by qRT-PCR, and the target genes were further explored by bioinformatics analysis. Correlation analyses were carried out to compare the expression of miRNA and target genes. Then, mutants, miRNA mimics and inhibitors of the target genes were constructed and transfected to colonic epithelial cells. qRT-PCR, western blotting, enzyme-linked immunosorbent assays (ELISAs) and dual-luciferase assays were used to investigate the expression of miR-144 and OCLN, ZO1 in IBS-D rats. Results: There were 8 up-regulated and 18 down-regulated miRNAs identified in the IBS-D rat model. Of these, miR-144 was markedly up-regulated and resulted in the down-regulation of OCLN and ZO1 expression. Overexpression of miR-144 by transfection of miR-144 precursor markedly inhibited the expression of OCLN and ZO1. Further studies confirmed that OCLN and ZO1 were direct targets of miR-144. Additionally, intestinal hyperpermeability was enhanced by miR-144 up-regulation and attenuated by miR-144 down-regulation in IBS-D rat colonic epithelial cells. Moreover, rescue experiments showed that overexpression of OCLN and ZO1 significantly eliminated the inhibitory effect of miR-144, which showed a stronger effect on the attenuation of intestinal hyperpermeability. Conclusion: Up-regulation of miR-144 could promote intestinal hyperpermeability and impair the protective effect of the epithelial barrier by directly targeting OCLN and ZO1. miR-144 is likely a key regulator of intestinal hyperpermeability and could be a potential therapeutic target for IBS-D.

MicroRNA-200a Targets Cannabinoid Receptor 1 and Serotonin Transporter to Increase Visceral Hyperalgesia in Diarrhea-predominant Irritable Bowel Syndrome Rats

“…Colonic Epithelial Cells (CECs) were isolated and cultured as described previously (Supplementary Material 6). 17,22 Bioinformatic Analysis MiRanda (http://www.microrna.org), PicTar (http://pictar. mdc-berlin.de), and targetScan (http://www.targetscan.org) were used in the bioinformatic analysis of miRNAs.…”

“…The method was described previously. 17 Briefly, CECs from the IBS-D rats and control rats were seeded into 6-well culture plates and transfected with RNA oligoribonucleotides (miR-200a mimic, miR-200a inhibitor, mimic control, inhibitor control, silent-CNR1 (si-CNR1), and silent-SERT (si-SERT)) for 6 hours, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). CECs from control rats transfected with nothing were treated as the control group, while CECs from IBS-D rats transfected with mimic control were treated as the model group.…”

“…[14][15][16] In our previous paper, we reported that the up-expression of miR-144 in the intestinal tissue of the IBS-D rat promotes intestinal permeability. 17 However, the role of miRNAs in regulating visceral hypersensitivity of IBS-D remains largely un-know.…”

DNA methylation, microRNA expression profiles and their relationships with transcriptome in grass-fed and grain-fed Angus Cattle rumen tissue

“…Additionally, two of the 145 target genes were found in the DEGs list, which were OCLN and RBM47, respectively. OCLN was a key tight junction protein that could interact with intracellular signaling pathways, which played roles in regulating intestinal function 50 . RBM47 was a novel RNA-binding protein; it could contribute to the basic machinery for C to U RNA editing in intestine, influencing the expression of some genes 51 ; however, the function of this gene has not been studied in rumen.…”

Deep-Fried Atractylodis Rhizoma Protects against Spleen Deficiency-Induced Diarrhea through Regulating Intestinal Inflammatory Response and Gut Microbiota

“…Studies have shown that the integrity of the intestinal barrier is mainly related to the normal activity of tight junction proteins. Previous studies have shown that the expression of ZO-1 and Occludin is significantly reduced in diarrheal diseases [15,16]. In addition, it has been found that PAR-2 and p38MAPK are involved in the regulation of tight junction protein expression [17].…”