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MiR-144 Increases Intestinal Permeability in IBS-D Rats by Targeting OCLN and ZO1
Qiuke Hou1, Yongquan Huang2, Shuilian Zhu1
1Department of Gastroenterology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China.
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.
Insights
In irritable bowel syndrome with diarrhoea (IBS-D), up-regulated miR-144 promotes intestinal hyperpermeability by targeting occludin (OCLN) and zonula occludens 1 (ZO1). Down-regulating miR-144 may restore the epithelial barrier, offering a potential therapeutic strategy for IBS-D.
Area of Science:
- Gastroenterology
- Molecular Biology
- MicroRNA Research
Background:
- Irritable bowel syndrome with diarrhoea (IBS-D) is linked to intestinal hyperpermeability.
- MicroRNAs (miRNAs) play a role in regulating intestinal barrier function, but their specific involvement in IBS-D remains unclear.
- This study investigated the role of miRNAs in regulating intestinal permeability and epithelial barrier integrity in IBS-D.
Purpose of the Study:
- Identify differentially expressed miRNAs and their targets in the distal colon of IBS-D rats.
- Verify if occludin (OCLN) and zonula occludens 1 (ZO1) are direct targets of miR-144 and are down-regulated in IBS-D.
- Determine if miR-144 down-regulation can reverse intestinal hyperpermeability by targeting OCLN and ZO1.
Main Methods:
- Established an IBS-D rat model using acetic acid.
- Performed miRNA microarray analysis and qRT-PCR on colonic tissues.
- Utilized bioinformatics, western blotting, ELISA, and dual-luciferase assays to analyze miRNA-target interactions.
- Transfected colonic epithelial cells with miRNA mimics and inhibitors.
Main Results:
- Identified 8 up-regulated and 18 down-regulated miRNAs in IBS-D rats.
- Found miR-144 significantly up-regulated, leading to down-regulated OCLN and ZO1 expression.
- Confirmed OCLN and ZO1 as direct targets of miR-144.
- Demonstrated that miR-144 up-regulation enhances intestinal hyperpermeability, while its down-regulation attenuates it.
Conclusions:
- Up-regulation of miR-144 promotes intestinal hyperpermeability and impairs the epithelial barrier by targeting OCLN and ZO1.
- miR-144 is a key regulator of intestinal hyperpermeability in IBS-D.
- miR-144 represents a potential therapeutic target for IBS-D.
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