Mechanism Of CircRNA＿103765 By Sponging MiR-30 Regulate DLL4/Notch Pathway In Crohn’s Disease

Part Ⅰ The differential expression of circRNAs in PBMCs of CD patientsObjective:The purpose of this study was to screen the differentially expressed circRNAs in peripheral blood mononuclear cells（PBMCs）of patients with CD and healthy control（HC）by circRNAs gene chip technology,and to find new biomarkers for the diagnosis and evaluation of CD.Methods:Five active CD patients and 5 healthy controls were selected for circRNAs microarray screening.At the same time,PBMCs samples from 60 patients with active CD and 40 healthy controls were collected.Then,the circRNA₁03765 in PBMCs was detected by reverse transcription polymerase chain reaction（qRT-PCR）.The diagnostic value circRNA₁03765 was evaluated by receiver operating characteristic（ROC）curve.Spearman’s analysis was used to analyze the linear correlation between circRNA₁03765 and clinical indexes（CRP、TNF-α、CDAI）.Results:384 circRNAs differentially expressed were found in the gene chip screening,155 up regulated and 229 down regulated.Further validation showed that the relative expression level of circRNA₁03765 in CD groups was significantly higher than that of HC group（P<0.05）.The CD curve（AUC）of circRNA₁03765 was 0.7015.In addition,in CD group,the relative expression of circRNA₁03765 was positively correlated with CRP（R2=0.2886,P<0.001）,TNF-α（R2=0.5122,P<0.001）and CDAI（R2=0.5625,P<0.001）.Conclusion:circRNA₁03765 in PBMCs of CD patients was significantly up regulated and could be used as a potential biomarker for CD diagnosis and evaluation.Part Ⅱ The detection of circRNA₁03765 in active CD patients treated with anti TNF-α monoclonal antibodyObjective:To evaluate the circRNA₁03765 of active CD patients before and after treatment with anti TNF-α monoclonal antibody.The purpose of this study was to investigate the correlation between the change of circRNA₁03765 and TNF-α in IBD patients.Methods:45 patients with active CD were treated with anti TNF-α monoclonal antibody（infliximab,IFX,5 mg/kg）.The clinical efficacy was evaluated after IFX treatment.Before IFX treatment and the fourth IFX infusion,circRNA₁03765 and TNF-α were detected.The expression of circRNA₁03765 was detected by reverse transcription polymerase chain reaction（qRT-PCR）,meanwhile,TNF-α was detected by CBA.The linear correlation ship between circRNA 103765 and TNF-α analyzed by Spearman linear regression.Results:21 CD patients achieved clinical remission（CDAI<150）,13 patients had clinical response（CDAI decrease≥70 but CDAI≥150）.IFX treatment failed in 11 CD patients（CDAI≥150 and CDAI decrease≤70）.The circRNA₁03765 expression in clinical remission group and response group after IFX treatment was significantly lower than that before IFX treatment（P<0.05）.However,the expression of circRNA₁03765 in the failure group was unchanged.The level of TNF-α in clinical remission group was significantly lower than that before IFX induction treatment（P<0.05）.However,no significant change of TNF-α was observed in the clinical response group and treatment failure group（P>0.05）.In addition,the TNF-α level was greatly positively correlated with circRNA₁03765 expression in the remission group after IFX therapy in CD patients,as well as before IFX induction therapy.Nevertheless,no important correlation between circRNA₁03765 and TNF-α was found in the response or failure groupConclusion:IFX treatment can effectively reduce the circRNA₁03765 in the clinical remission group.The expression changes of circRNA₁03765 and TNF-α were positively correlated.Part Ⅲ TNF-α induces circRNA₁03765 transcription and its effect on cell proliferation and apoptosis in a human intestinal epithelial cell lineObjective:To investigate TNF-α induces circRNA₁03765 transcription by effecting cell proliferation and apoptosis in a human intestinal epithelial cell lineMethods:The blank control（Mork group）,TNF-α stimulated intestinal epithelial cell lines（Caco-2 and HIEC）,z-VAD-FMK pretreatment group and small interfering RNA（sicircRNA₁03765）group were set up.The expression of circRNA₁03765 was detected by qRT-PCR.CCK-8 was used to detect the proliferation of TNF-α stimulated cells.Annexin V/PI staining was used to detect cell apoptosis.Western blot was used to detect the expression of Bax,Bcl-2 and cleaved caspase-3.Results:Compared with the blank control group,TNF-α stimulated circRNA₁03765 expression in a dose-and time-dependent manner.The expression level of circRNA₁03765 was considerable upregulated in Caco2 and HIECs compared with that in the controls after exposure to TNF-α（P<0.001）.Flow cytometry-based Annexin V/PI staining showed that TNF-α significantly increased the apoptotic rate compared with the negative control（P<0.05）.Furthermore,TNF-α increased the levels of the proapoptotic proteins Bax and cleaved caspase-3 and decreased the expression of Bcl-2,In addition,we pretreated Caco2 and HIEC cells with broad-spectrum caspase inhibitor z-VAD-FMK,and then induced with TNF-α.The results showed that z-VAD-FMK induced suppression of circRNA₁03765 expression in a concentration-and time-dependent manner.Small interfering RNA（si_circ₁03765）was used to knock down circRNA₁03765 in a TNF-α-treated Caco2 and HIEC.qRT-PCR showed that the siRNA greatly decreased the expression level of circRNA₁03765（P<0.05）.And downregulated circRNA₁03765 significantly promoted the proliferation of Caco2 cells and HIEC cells（P<0.05）.Simultaneously,the cell apoptosis rate and the expression of apoptosis related proteins were significantly decreased in siRNA intervention group（P<0.05）.Conclusion:The expression of circRNA₁03765 was up-regulated in human intestinal epithelial cell line which stimulated by TNF-α.Inhibition of apoptosis can down regulate circRNA₁03765 expression.And si-circRNA₁03765 can down regulates the expression of circRNA₁03765 and alleviate cell apoptosis and promote cell proliferation what TNF-αinduced.Part Ⅵ CircRNA₁03765 promotes human intestinal epithelial cell proliferation and apoptosis through the circRNA₁03765/miR-30/DLL4/Notch signaling axisObjective:To explore that circRNA₁03765 can act as a competitive endogenous RNA（ceRNA）through the circRNA₁03765/miR-30/DLL4 axis.Methods:The TargetScan and miRanda databases were used to predict the targets of circRNA₁03765.QRT-PCR was used to detect the expression of miR-30 in PBMCs of CD patients before and after treatment with anti TNF-α monoclonal antibody.A dual luciferase reporter assay and fluorescence in situ hybridization（FISH）were used to validate the sponging relationship between circRNA₁03765 and miR-30a-5p and 30e-5p in 293T cells.After transfection with miR-30a-5p/miR-30e-5p mimics and inhibitors,the expression of circRNA₁03765 was detected by qRT-PCR.CCK-8 was used to detect the cell proliferation.Annexin V/PI staining was used to detect cell apoptosis.Western blot was used to detect the expression of Bax,Bcl-2,cleaved caspase-3,DLL4,NICD and Hes1.Results:The TargetScan and miRanda databases showed that circRNA₁03765 contains a conserved target site of the miR-30 family（miR-30a-5p,miR-30b-5p,miR-30d5p,miR-30e-5p）.The expression level of circRNA₁03765 was negatively correlated with miR-30a-5p and miR-30e-5p expression in PBMCs from CD patients prior to and after treatment with IFX（P<0.001）.However,there was no significant correlation between circRNA₁03765 and miR-30b-5p or miR-30d-5p.FISH showed circRNA₁03765（red）and miR-30a-5p/30e-5p（green）were mainly colocalized in the cytoplasm.DLL4 is a target gene of miR-30.In addition,transfection of miR-30a-5p/miR-30e-5p mimics could rescue the proliferation and apoptosis induced by TNF-α.On the contrary,miR-30a-5p inhibitors or miR-30e-5p inhibitors reversed the proliferation and apoptosis effects of circRNA₁03765 knock-down in Caco2 cells and HIECs.In addition,TNF-α-induced upregulation of circRNA₁03765 enhanced the protein and mRNA levels of DLL4 and the downstream proteins NICD and Hes1,while downregulated circRNA₁03765 decreased the levels of these proteins.Conclusion:circRNA₁03765 might serve as a ceRNA for miR-30a-5p/miR-30e-5p to regulate the expression level of DLL4...