MiR-124 Targeting AHR In Intestinal Epithelial Cells And Is Associated With The Pathogenesis Of Crohn's Disease

ObjectiveEnvironmental factors are believed to contribute to the increased prevalence of inflammatory bowel disease(IBD).The aryl hydrocarbon receptor(AHR)modulates the activity of immune and nonimmune cells in the gut,and is involved in environmental factors regulating mucosal immune responses.Recent studies showed that microRNAs(miRNAs)regulate gene expression in innate immune cells such as intestinal epithelia cells(IECs).Dysregulation of miRNAs is associated with some human autoirnmune diseases including Crohn's disease(CD),but the mechanisms remain largely unknown.In the present study,we investigated the contribution of AHR expression-associated miRNAs to modulate disruption of colonic epithelial cells and is associated with the pathogenesis of Crohn's Disease.Methods:1·Levels of AHR expressions in colon tissues from active CD patients and healthy controls were assessed by real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR),western blotting and immunohistochemistry(IHC).2.Levels of inflammation-related miRNAs in colon tissues from active CD patients and healthy control were assessed by qRT-PCR.MIRNA-AHR pairs was predicted by Targetscan Human 6.2.3.Level of sequence complementarity between miRNA and target site of AHR 3'UTR was validated by luciferase reporter assay.4.The expression of AHR protein and mRNA were detected by western blotting and qRT-PCR after overexpression or knockdown of miRNA in Caco-2 cells.5.Caco-2 cells were stimulated with LPS after overexpression or knockdown of miRNA.The levels of pro-inflammatory cytokine in culture supernatants were measured by enzyme linked immunosorbent assay(ELISA).Results:1.While AHR protein levels were significantly decreased in colon tissues of active CD patients,AHR mRNA varied randomly between active CD tissues and normal controls.2.MiR-124 expression was significantly increased in these inflammation-related miRNAs,which had an inverse correlation with AHR protein expression.Bioinformatics prediction showed that AHR was the target gene of miR-124.3.The luciferase reporter assay confirmed that miR-124 directly combined with the 3'UTR region of AHR.4.In vitro,over-expression of miR-124 led to AHR expression decreased,while knockdown of miR-124 resulted in AHR expression increased.And AHR mRNA expression did not change.5.Over-expression of miR-124 in Caco-2 cells increased the expression of LPS-induced pro-inflammatory cytokine by suppressing AHR expression.Conclusion:These findings suggest that miR-124 regulate inflammatory development by suppresses its target AHR and is involved in the pathogenesis of CD.