| Inflammatory bowel disease(IBD) including ulcerative colitis(UC) and Crohn’s disease(CD), is a chronic relapsing inflammatory disorder of the intestines. Patients with UC may present with frequent stool, rectal bleeding, lower abdominal pain, tenesmus, whereas CD present with abdominal pain, diarrhea, weight loss, and fever. So far, the pathogenesis of IBD remains unclear, emerging investigation have shown that genetic predisposition,environmental factors, immune disorders contribute to the development of IBD. mi RNA is a class of evolutionarily conserved single-stranded and non-coding RNA molecules, and recent studies have shown that mi RNA play an important role in the development of many diseases. Emerging research indicate that mi RNA can regulate Th17 cells differentiation and function in multiple sclerosis, systemic lupus erythematosus and other autoimmune diseases. However, how mi R-21 regulates immune responses in gut mucosa, particularly in inflammatory bowel disease(IBD), remains to be elucidated.Aims: To explore the level of mi R-21 in the CD4~+ T cells from peripheral blood mononuclear cells(PBMC) and inflamed gut mucosa of IBD patients and further investigate the role and significance of mi R-21 in the pathogenesis of IBD.Method: Colon mucosa tissues and PBMC were obtained from 41 patients with active IBD,32 IBD patients in remission and 24 healthy controls, and the level of mi R-21 in these samples were analyzed by q RT-PCR. CD4~+ T cells, CD8+ T cells, B cells, dendritic cells,neutrophils, monocytes and intestinal epithelial cells were isolated from peripheral blood mononuclear cells and colon mucosal biopsies by using Magnetic beads. mi R-21 expression in each subsets of immune cells were detected by q RT-PCR. CD4~+ T cells were isolated from the PBMC and inflamed intestinal mucosa, respectively and expression of mi R-21 were detected by q RT-PCR. CD4~+ T cells were separated from patients with IBD and then stimulated with TNF-a,(40)L-1,IL-6,IL-17 for 48 h, respectively and expression of mi R-21 in each group was measured by q RT-PCR. PBMC and gut mucosa were collected from CD patients before and after IFX treatment and expression of mi R-21 was measured by q RT-PCR. CD4~+ T cells isolated from patients with IBD and healthy donors were cultured in medium and transfected with LV-mi R-21, LV-anti-mi R-21 and LV-mi R-control lentivirus for 72 h, respectively, and then levels of mi R-21, IL-17 A, TNF-a and IFN-g were measured by q RT-PCR and ELISA.Results: mi R-21 expression in PBMC and colonic biopsies obtained from patients with active IBD were significantly higher than that in healthy controls, and CD4~+ T cells were an important source of mi R-21. Further investigation indicated that expression of mi R-21 in CD4~+ T cell is markedly increased in IBD patients compared with healthy controls. In vitro,we found that mi R-21 expression was significantly increased after TNF-a treatment and IL-6 modestly enhanced mi R-21 expression in CD4~+ T cell, while IL-17 A, IFN-g, IL-23 had no effect on mi R-21 expression. Moreover, expression of mi R-21 is markedly decreased in CD4~+ T cell isolated from CD patients after IFX therapy. mi R-21 expression was significantly increased in LV-mi R-21 transduction cells and it was markedly down-regulated in cells transduced with LV-anti-mi R-21 compared with LV-controls.m RNA levels of RORC, IL-17 A, TNF-a were found to be significantly increased in CD4~+T cells transfected with LV-mi R-21. Consistently, the protein levels of TNF-a and IL-17 A were markedly up-regulated in the supernatants of CD4~+ T cells transfected with LV-mi R-21 compared with controls, whereas, IFN-g, T-bet, IL-4, Gata-3 and Foxp3 did not alter markedly.Conclusions: mi R-21 expression is significantly up-regulated in IBD CD4~+ T cells both from PBMC and inflamed gut mucosa. Disturbing expression of mi R-21 may be resulted from, at least partly, the increased level of TNF-g in patients with IBD. mi R-21 may promotes the development of IBD through inducing Th17 cell differentiation and IL-17 A and TNF-a production. These result has shed a new light on understanding the pathogenic role of mi R-21 in the pathogenesis of IBD and suggests that target therapy directly against mi R-21 may be a novel approach for treatment of patients with IBD. |
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