Expression Of MicroRNA-21 In Inflammatory Bowel Disease And Its Regulatory Effect On CD4~+T Cell Differentiation

Objective:Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.IBD has a long course,is prone to recurrence,and has many complications,bringing a huge burden of disease to patients and the medical system.It is important to explore the pathogenesis and intervention targets of IBD and then find new drugs and treatment strategies.Studies have found that micro RNA(miRNA)is associated with the pathogenesis of IBD,of which micro RNA-21(miR-21)is one of the most studied miRNAs in IBD.CD4~+T cells play a key role in the pathophysiology of IBD,while miR-21 is actively involved in T cell differentiation,proliferation,development,and apoptosis.Whether miR-21 is involved in the pathogenesis of IBD by regulating CD4~+T cell differentiation has not been well studied.This study intends to elucidate the relationship between miR-21 and IBD and the molecular mechanism by which miR-21 may regulate CD4~+T cell differentiation,so as to provide a basis for screening novel IBD markers and provide a new intervention site for the prevention and treatment of IBD.Methods:In this study,clinical samples from IBD patients were collected and a mouse model of Dextran sulfate(DSS)-induced acute colitis was constructed.RT-qPCR,ELISA,WB,fluorescence in situ hybridization and flow cytometry were used to deeply explore the expression level of miR-21 and the percentage change of CD4~+T cell subsets in human and mouse colon inflammation.Na(?)ve CD4~+T cells are sorted by magnetic beads from mouse spleen.The expression level of miR-21 was then regulated by transfecting miR-21 inhibitor/mimics or corresponding NC into cells.The transfected Na(?)ve CD4~+T cells were induced to differentiate in the Th1,Th2,Th17 and Treg directions,respectively.Flow cytometry,RT-qPCR,and ELISA were used to observe the effect of miR-21expression changes on CD4~+T cell differentiation.Database screening and dual-luciferase reporter assay system were used to find target genes for miR-21 and elucidate the molecular mechanism by which miR-21 regulates CD4~+T cell differentiation in IBD.Therapeutic intervention in mice with DSS-induced acute colitis was attempted by injecting miR-21 antagomir into mice.Results:(1)In the patient’s intestinal mucosal tissue,mouse colon tissue and spleen lymphocytes,the expression level of miR-21 was significantly increased in the inflammatory group compared with the normal control group.Compared with the control group,the expression levels of the characteristic transcription factors T-BET,GATA3 and RORγt of Th1,Th2 and Th17 cells were increased in the colon and spleen lymphocytes of mice with DSS-induced acute colitis,while the expression levels of the characteristic transcription factor Foxp3 in Treg cells were relatively reduced.Compared with the control group,in spleen lymphocytes of mice with DSS-induced acute colitis,the percentages of Th1(4.01±0.24 VS 6.47±0.16,P<0.001),Th2(3.88±0.47 VS 5.64±0.32,P<0.05)and Th17(8.00±0.29 VS 12.83±0.87,P<0.01)increased significantly,while the percentage of Treg decreased significantly(46.23±1.88 VS 24.33±3.97,P<0.01).(2)The elevated expression level of miR-21 had the most significant effect on the differentiation of Na(?)ve CD4~+T cells in the direction of Th17 cells.The increase in miR-21 expression levels promoted the differentiation of Na(?)ve CD4~+T cells in the Th2and Th17 directions,and inhibited their differentiation in the Th1 and Treg directions.(3)Two antagonists of the TGF-βsignaling pathway,Smad7 and Ski,were target genes of miR-21.By downregulating Smad7 and Ski,miR-21 promoted the differentiation of Na(?)ve CD4~+T cells in the direction of Th17 and inhibited their differentiation in the direction of Treg.Both Smad7 and Ski were able to eliminate the effect of miR-21 on the regulation of Th17 and Treg differentiation.(4)miR-21 antagomir was able to reduce the expression level of miR-21 and inhibit the Phospho-(p-)Smad2/3 signaling pathway.It reduced the proportion of Th17 cells,increased the proportion of Treg cells,improved symptoms and relieved tissue inflammation in mice with DSS-induced acute colitis by upregulating Smad7 and Ski.Conclusions:In IBD,miR-21 promotes Th17 cell differentiation and inhibits Treg cell differentiation by inhibiting the expression of Smad7 and Ski,and then activating the p-Smad2/3 signaling pathway,thus promoting intestinal inflammation.