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.
Background/AimsMicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown.MethodsWe established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats.ResultsThe IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs.ConclusionsThis study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.
It has been previously reported that psoralen, one of the active ingredients in Psoralea corylifolia, could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), suggesting its potential to treat osteoporosis. Additionally, runt-related transcription factor 2 (Runx2) is a transcription factor that plays vital roles in BMSC osteogenic differentiation. However, whether and how microRNAs (miRNAs/miRs) modulate osteogenic differentiation induced by psoralen have not yet been examined, to the best of the authors' knowledge. The present study aimed to identify the miRNA target genes that regulate osteogenic differentiation of BMSCs induced by psoralen. A Cell Counting Kit-8 assay and alizarin red staining were used to detect the viability and osteogenic differentiation of BMSCs, respectively, under treatment with psoralen. miRNA microarray analysis was performed to identify the differentially expressed miRNAs under treatment with psoralen. A bioinformatics analysis and a luciferase reporter assay were conducted to identify the targets of miR-488. Finally, the mechanisms of miR-488 in psoralen-induced BMSC osteogenic differentiation were investigated using overexpression or inhibition methods in vitro. Cell viability was elevated and osteogenic differentiation of BMSCs was improved under treatment with psoralen. miRNA microarray analysis and further validation by reverse transcription-quantitative PCR revealed that miR-488 was downregulated during psoralen-induced BMSC osteogenic differentiation. Bioinformatics analysis and experimental validation by a luciferase reporter assay identified Runx2 as a potential target of miR-488. Overexpression of miR-488 by transfection with miR-488 mimics markedly inhibited the expression of Runx2, Osterix and alkaline phosphatase, whereas, the inhibition of miR-488 expression by the miR-488 inhibitor promoted their expression compared with the control. Rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR-488 on BMSC osteogenic differentiation. The present results suggested that miR-488 is a negative regulator of psoralen-induced BMSC osteogenic differentiation by targeting Runx2, providing a possible therapeutic target for osteoporosis.
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