Dysregulated Circ?0001021 Involved In The Epithelial Barrier Function By Targeting Mir-224-5p In Ulcerative Colitis

BackgroundUlcerative colitis(UC)is one form of the inflammatory bowel disease(IBD)within colorectum.Inflammation is characteristically limited to the mucosal surface with cryptitis and crypt abscesses.UC usually presents with diarrhea,often with blood or pus.It is diagnosed by colonoscopy and histological findings.According to research statistics,the incidence of UC is rising rapidly,and the global prevalence rate exceeds 0.3%.Currently,mucosal healing is considered as an important therapeutic goal.However,commonly prescribed therapies show adverse effects and fail to achieve treatment goals.The pathogenesis of UC is not fully understood.Understanding UC pathogenesis will contribute to develop a better therapeutic approach and ease disease burden.Comprehensive epidemiologic and genetic studies suggested that UC was arising from an abnormal interaction between genetics,immune dysregulation and environmental factors.These factors lead to disorders of the mucosal immune,microbial dysbiosis and defective intestinal barriers.In particular,the intestinal epithelial barrier serves as the first physical and immune protective wall to resist the invasion of toxins and pathogens.Excessive death of intestinal epithelial cells and changes in the expression and distribution of tight junction proteins can cause damage to the intestinal epithelial barrier.Recently,non-coding RNAs(ncRNAs)expression levels have been reported to be dysregulated in UC patients and played important roles in regulating the epigenetics of UC.Genome Wide Association Study have shown that UC-associated risk loci appear to affect ncRNAs.NcRNAs are involved in UC by regulating gene expression at transcriptional and translational levels.Circular RNAs(circRNAs)belong to the family of ncRNAs.The differential expression of circRNAs was reported in UC patients.In addition,circRNAs are related to the inflammatory response of UC,such as cytokine and chemokine regulation,autophagy disorders and intestinal epithelial barrier regulation.However,the roles of circRNAs in UC are still not fully elucidated.Therefore,unraveling the mechanisms of the key circRNAs affecting the intestinal epithelial barrier has profound clinical implications for UC prevention and therapeutic strategies.This study was divided into three parts to explore role of circRNAs in intestinal epithelial barrier,including to establish the circRNAs expression profile based on the whole-transcriptome sequencing of UC mucosal biopsy;to identify the circRNA that is significantly related to UC,and exploring the roles in UC;to validate the mechanism of circRNAs participated in the modulation of intestinal epithelial barrier function in UC.Part ?:Characteristic changes of whole-transcriptome sequencing in patients with ulcerative colitisAims:CircRNAs performed diverse biological functions and were important regulators multiple diseases.However,few circRNAs have been thoroughly explored in UC.Therefore,whole-transcriptome sequencing on mucosa sample of UC patients were performed and the circRNAs,miRNAs and mRNAs expression profiles were established.Methods:Patients with active UC and healthy controls(HCs)aged 20-65 years were recruited from Qilu hospital of Shandong university.Mayo score and histological score was used to assess UC severity.Collected colonic mucosa biopsy samples from UC patients and HCs for circRNAs,mRNAs microarrays and miRNAs sequencing.The circRNAs,miRNAs and mRNAs expression profiles were established based on bioinformatics analysis.CircRNAs target genes were predicted and circRNAsmiRNAs networks were constructed,followed by the functional enrichment.Results:A total of 30 UC patients and 25 HCs were recruited.Demographic indicators were well balanced between UC patients and HCs.5 paired inflamed and non-inflamed samples from UC patients and 5 HCs samples were used to compare profile of circRNAs,miRNAs and mRNAs.Correlation analysis showed that the gene expression levels among all test samples were highly correlated.The results indicated high experimental reliability and reasonable sample selection.A total of 11,338 circRNAs(7,272 upregulated and 4,066 downregulated),321 miRNAs(159 upregulated and 162 downregulated)and 2,149 mRNAs(1,183 upregulated and 966 downregulated)were differentially expressed in inflamed UC colonic mucosa compared with HCs colonic mucosa.A total of 7,261 different expression circRNAs(4,059 upregulated and 3,202 downregulated),169 different expression miRNAs(67 upregulated and 102 downregulated)and 1,480 different expression mRNAs(706 upregulated and 774 downregulated)were identified between non-inflamed UC colonic mucosa and HCs colonic mucosa.Subsequently,intersection of different expression circRNAs and miRNAs from the comparison non-inflamed UC colonic mucosa vs HCs colonic mucosa as well as the comparison inflamed UC colonic mucosa vs HCs colonic mucosa were conducted.The shared differential ncRNAs expression with same change were identified,including 580 dysregulated circRNAs,87 dysregulated miRNAs.Following,the circRNAs-miRNAs regulatory networks were constructed,including 28 circRNAs-miRNAs pairs.Functional enrichment analysis showed acute inflammatory response,response to IL-1,cellular response to biotic stimulus,complement and coagulation cascade,and HIF-1 signaling pathway were participated in UC.Conclusions:Obvious differential expression of circRNAs were observed in UC patients.The differential expression circRNAs may be involved in the occurrence and development of UC.Part ?:Hsa_circ₀001021 regulated intestinal epithelial barrier function in ulcerative colitisAims:The intestinal epithelial barrier plays an essential role in UC.According to the circRNAs-miRNAs networks of Part I,hsa_circ₀001021 was downregulated in UC.Based on the circBase database,hsa_circ₀001021 is derived from human AFTPH in chr2.AFTPH was reported to be expressed in colonic epithelial cells.AFTPH level was downregulated in UC patients and in the mice with TNBS-induced and DSSinduced colitis.Due to the same source and location between hsa_circ₀001021 and AFTPH,hsa_circ₀001021 was thought might to be involved in the pathogenesis of UC.Thus,colonic biopsy samples and colon epithelial cell lines were employed to explore the roles of hsa_circ₀001021 in UC.Methods:1.The bioinformatics analysis of hsa_circ₀001021:Bioinformatics analysis was performed to determine hsa_circ₀001021 location,nucleotide length,the ability of coding protein and binding miRNA.2.The expression level of hsa_circ₀001021 in UC patients:The expression of hsa_circ₀001021 was verified in colonic mucosa samples of 25 UC patients and 20 HCs using qRT-PCR.Detailed sample information,including Mayo score,Mayo endoscope score,histological score,serum inflammatory index(ESR)of each patient were collected to evaluate the clinical significance of hsa_circ₀001021.3.The effects of hsa_circ₀001021 on intestinal epithelial barrier function1)Fluorescence in situ hybridization(FISH)was performed to determine the location of hsa_circ₀001021 in intestinal mucosa.The correlation between hsa_circ₀001021 expression and tight junction protein was detected by clinical samples.2)The effect of hsa_circ₀001021 on intestinal epithelial barrier permeability and tight junction protein expression was detected by cell transfection experiments,fluorescein isothiocyanate-dextran(FD4)permeability assay and western blotting analysis.Results:1.Hsa_circ₀001021 contained 1967 nucleotides and was derived from the exon 2 and 3 of human AFTPH in chr2.It had miRNA response element(MRE),RNA binding protein(RBP)and open reading frame(ORF).2.Abnormal hsa_circ₀001021 expression was correlated with clinical characteristics in UC patients:Compared with HCs colonic mucosa,hsa_circ₀001021 was downregulated in inflamed UC colonic mucosa and non-inflamed UC colonic mucosa.In addition,the expression level of hsa_circ₀001021 was negatively correlated with Mayo score,histological score and serum ESR.3.Hsa circ 0001021 regulated intestinal epithelial barrier function1)FISH staining showed that hsa_circ₀001021 was located in intestinal epithelial cells.The expression level of hsa_circ₀001021 was positively related to tight junction proteins ZO-1 and occludin,and negatively correlated with CLDN-2.2)Paracellular permeability was increased in HT-29 and caco2 cells with sihsa_circ₀001021 transfection.Besides,decreased expression of ZO-1 and occludin were observed in HT-29 and caco2 cells with knockdown of hsa_circ₀001021,as well as increased expression of CLDN-2.Conclusions:Hsa_circ₀001021 was downregulated in UC patients and related to UC severity.hsa_circ₀001021 could modulate intestinal epithelial barrier function by regulating tight junction expression.Part ?:The mechanism of dysregulated hsa_circ₀001021 involved in the epithelial barrier function by targeting miR-224-5p in ulcerative colitisAims:CircRNAs are known to have multiple functions,which include serving as miRNA sponges,interacting with RBP,modulating alternative splicing and transcription,translation.Based on the Part ? the study,hsa_circ₀001021 was derived from exon and had MRE.FISH staining showed that hsa_circ₀001021 was located in the cytoplasm of intestinal epithelial cells.Most circRNA located in the cytoplasm played the role by binding miRNA to regulate the expression of miRNA target gene.Hsa_circ₀001021 was thought might to be regulate intestinal epithelial barrier function through competitive endogenous RNA(ceRNA)mechanism.Thus,colonic biopsy samples and colon epithelial cell lines were employed to validate the mechanism of hsa_circ₀001021 involved in epithelial barrier function in UC.Methods:1.Based on circRNAs-miRNAs network of Part ?,the combination of hsa_circ₀001021 and miR-224-5p was verified.Bioinformatics analysis was performed to predict miR-224-5p target genes smad4.2.The expression of miR-224-5p and its correlation with UC severity were detected.The correlation between miR-224-5p expression level and tight junction protein expression were also detected.The expression of smad4 and its correlation with UC severity were detected.The correlation between smad4 expression level and tight junction protein expression were also detected.3.The luciferase experiments were performed to identify the combination between hsa_circ₀001021 and miR-224-5p,as well as between miR-224-5p and smad4.4.Western blotting analysis were performed to detect the effect of miR-224-5p on smad4 and tight junction protein expression in intestinal epithelial cells after miR224-5p overexpression or miR-224-5p inhibitor in intestinal epithelial cells.5.MiR-224-5p inhibitor was added into intestinal epithelial cells that interfered with si-hsa_circ₀001021,and the salvage experiment was conducted to observe whether miR-224-5p inhibition could reverse the hsa_circ₀001021-induced abnormal expression of samd4 and epithelial tight junction protein.Results:1.According to the circRNAs-miRNAs networks of Part I,hsa_circ₀001021 can bond to miR-224-5p.Bioinformatics analysis and previous research showed that miR224-5p may be combined with smad4.2.Compared with HCs colonic mucosa,miR-224-5p was upregulated in inflamed UC colonic mucosa and non-inflamed UC colonic mucosa.The expression level of miR224-5p was positively correlated with Mayo score.MiR-224-5p expression level was negatively related to ZO-1 and occludin,and positively correlated with CLDN-2.In clinical samples,the expression level of hsa_circ₀001021 were negatively correlated with miR-224-5p.Moreover,compared with HCs colonic mucosa,smad4 was downregulated in inflamed UC colonic mucosa and non-inflamed UC colonic mucosa.Smad4 expression level was negatively related to Mayo score.Smad4 expression level was positively associated with ZO-1 and occludin,and negatively correlated with CLDN-2.The expression level of hsa_circ₀001021 is positively correlated with smad4.3.Luciferase experiments showed that miR-224-5p bonded to hsa_circ₀001021,as well as smad4.4.The expression of smad4,ZO-1 and occludin were decreased and the expression of CLDN-2 was increased in HT-29 and caco2 cells with overexpression of miR-224-5p.The expression of smad4,ZO-1 and occludin were increased and the expression of CLDN-2 was decreased in HT-29 and caco2 cells with miR-224-5p inhibitors transfection.5.Rescue experiment showed miR-224-5p inhibitors reversed the effect of sihsa_circ₀001021 on downregulation of smad4,ZO-1 and occludin expression.Conclusions:Hsa_circ₀001021 could regulate the expression of smad4 via sponging miR-224-5p and modulate intestinal epithelial barrier function in UC.