Effects Of MiR-939 And MiR-376A On The Pathogenesis Of Ulcerative Colitis By Regulating NF-κB And NFAT Expression Through Decoy Strategy

BackgroundUlcerative colitis(UC)is a type of chronic,non-specific inflammatory lesions that mainly occur in the colorectal mucosa and submucosa,and presents intestinal mucosal hyperemia,erosion,ulcers,and hyperplasia[1].due to the complex etiology of UC and the coexistence of multiple pathogenesis,the clinical curative effect is not obvious and difficult to cure.The pathogenesis of UC mainly includes four aspects:susceptibility gene,intestinal barrier,cell cycle(apoptosis),immunity and inflammation[9-10]Therefore,it is of great significance to explore a fundamental and effective treatment method for various pathogenesis of UC to prevent and control the malignant transformation of UC and improve the quality of life of patients.Transcription factors(TFs)are a class of nuclear proteins that play a key positive or negative role in the regulation of gene expression.TFs almost control cell differentiation and organ formation,cell growth and apoptosis,and cell signal transduction by transcriptionally regulating gene expression.Transcription factors include nuclear transcription factor kappa B(NF-κB)and nuclear factor of activated T cells(NFAT).NF-κB and NFAT are involved in many biological processes such as immune response,inflammatory response,cell apoptosis and tumorigenesis[23-26].MicroRNAs(miRNAs)are small single-stranded RNA with a size of about 21-23 bases,which can participate in the regulation of gene expression.Recent studies have shown that miRNAs play an important role in the pathogenesis of UC[28-32].A new non-classical transcriptional regulation mechanism and function of miRNAs have been discovered in previous experimental studies.miR-939 and miR-376a are endogenous "decoy" of TFs,by inducing the transcription factor NF-κB and NFAT,thus regulating gene expression at the transcriptional level[8]This study aims to explore the specific mechanism of this decoy strategy in the occurrence and development of UC,and observe the therapeutic effect of decoy strategy on UC model mice.not only can this project supply the theoretical basis of the mechanism of UC pathogenesis,but also provide new ideas for clinical development of UC in the future.Objectives1.Verified the differential expression of miR-939 and miR-376a in mouse UC model by miRNA microarray analysis.And predict the target genes by TargetScan software.2.Investigate the expression of miRNA(-939,-3 76a)and TFs(NF-κB and NFAT)in the tissues of UC patients,and clarify the role of miR-939 and miR-376a in the pathogenesis of UC.3.Explore the specific mechanism of miRNAs’(-939,376a)decoy strategy for NF-κB and NFAT in the pathogenesis of ulcerative colitis.Study the effects of miR-939 and miR-376a on the pathogenesis of ulcerative colitis by regulating the expression of NF-κB and NFAT through decoy strategies.4.Verify the decoy strategy can treat UC model mouse.Methods1.Establish mouse UC model group and control group,and identify the differential expression of miRNA by miRNA microarray analysis.We chose miR-939 and miR-376a to have further investigation.Predicted the potential gene targets of miR-939 and miR-376a by TargetScan software.2.Collected the clinical samples of UC in human,and analyse the expression differences of miR-939,miR-376a,NF-κB,NFAT in tissue of normal samples and tissue of UC patients by QPCR,ISH and IHC.3.Establish the UC model cells in vitro.Experiment included seven groups via lipofectamine:miR-939 group,miR-939 inhibitor group,miR-376a group,miR-376a inhibitor group,Artificial NF-κB decoy ODN group,Artificial NFAT decoy ODN group and control group.Verify miRNAs’ decoy strategy for NF-κB and NFAT by Reporter Gene Assays.Detected the expression of related inflammatory factors(IL-8,IL-1β,IL-2,G-CSF,GM-CSF and MIP-2α)by ELISA.Detected the expression of immune factor(TNF-α and TLR4)by western blot.Observe the distribution of claudins,occludins,and ZO-1 by fluorescence microscopy.Detected the expression of claudins,occludins,and ZO-1 by QPCR and western blot.Detected the expression of cIAP,XIAP,Bax and Caspase-3 by western blot.Detected the level of apoptosis by flow cytometry.And observe the changes of apoptotic nucleus by fluorescence microscope.4.Establish the UC model mouse,Experiment included four groups via lipofectamine:Artificial decoy(NF-κB and NFAT)group,miRNAs(-939,-376a)decoy ODN group,UC model group and normal control group.Observe the changes of colon histomorphology in each group by HE staining.Detection of intestinal permeability in vivo by FITC-Dextran.Detected the expression of NF-κB p65 by IHC.Detected the expression of cIAP,XIAP,Bax and Caspase-3 by western blot.Detected the expression of related inflammatory factors(IL-6,IL-8,IL-1β and TNF-α)by ELISA.Analyze the therapeutic effect of miRNAs decoy strategy on UC mouse.Results1.Bioinformatics analysis revealed that miR-939 and miR-376a did not bind to the downstream genes of TFs(bcl-xl,FasL and TNF-α).2.The expressions of miR-939 and miR-376a in tissue of UC patients were significantly lower than that in normal tissues,and the expressions of NF-κB and NFAT in tissue of UC patients were significantly higher than that in normal tissues.In situ hybridization and immunohistochemical correlation analysis suggested that miR-939 was negatively correlated with NF-κB in tissue of UC patients and normal subjects,and miR-376a was negatively correlated with NFAT in tissue of UC patients and normal subjects.3.The results of luciferase reporter assay confirmed that miR-939 and miR-376a did have a "decoy" stratage for NF-κB and NFAT.And the "decoy" strategy reduced the expression level of related inflammatory factors,decreased the expression level of immune factors,increased the expression level of tight junction proteins.4.Animal experiments found that artificial decoy(NF-κB and NFAT)group and the transfected miRNAs(-93 9,-376a)decoy ODN group compared with UC model group,had A milder inflammatory response in the intestinal,Lower level of intestinal permeability,lower express of staining intensity of NF-KBp65 in colonic mucosal cells,lower express of apoptosis related proteins and lower express of related inflammatory factors.Conclusions1.The expression of miR-939 and miR-376a is reduced in tissue of UC patients.The expressions of miR-939 and NF-κB was negatively correlated in tissue of UC patients and tissue of normal subjects,and the expressions of miR-376a and NFAT was negatively correlated in tissue of UC patients and tissue of normal subjects.2.miR-939 and miR-376a did have a "decoy" stratage for NF-κB and NFAT,which can inhibite the expression of downstream inflammatory and immune related target genes,improve the expression level of the compact junction protein to repair the intestinal mucosal barrier and inhibiting the apoptosis of intestinal epithelial cells.3.The decoy strategy can inhibit the apoptosis of intestinal epithelial cells in mouse,reduce the secretion of inflammatory factors,repair the intestinal mucosal barrier,and has a certain therapeutic effect on UC model mouse.