| Rheumatoid arthritis(RA)is a chronic autoimmune disease characterised by synovial inflammation and a progressive cell-mediated destruction of joints,eventually leading to joints deformity and disability.Approximately 1% of the world’s population is afflicted by RA,with women being affected three times more often than men.Although various types of innate and adaptive immune cells have been shown to coordinately contribute to the pathogenesis of RA,data obtained from human and mouse studies suggested the central roles of CD4+ T cells.Epigenetics is a novel area of research in regulating cell physiology.As one kind of epigenetic modification,micro RNAs(mi RNAs),involved in a series of important physiological and pathological process,such as cell proliferation,apoptosis,differentiation,energy metabolism and immune regulation,display important roles in the pathogenesis of various diseases.mi RNAs are endogenous,small(21-25 nucleotides),non-coding RNAs that mediate m RNA cleavage and translational repression.Previous reports manifested that mi R-181 a had key roles in governing CD4+ T cell sensitivity,like mi R-181 a can influence the mature T cell activation threshold by T cell antigen receptor.And professor Goronzy found age is a critical factor of mi R-181 a expression.The expression of mi R-181 a declined with age,inhibited ERK signal pathway after TCR stimulation impairing T cell activation.A recent study exhibited that mi R-181 a is a key player in regulating inflammatory responses in vitro.However,it remains elusive that whether the mi R-181 a expresses higher or lower and how mi R-181 a is invovlved in the pathogenesis of RA.According to the literature,aberrant mi RNA expression has been detected in various cell types of RA,and these mi RNAs modulate specific pathways involved in RA pathogenesis.In this study,we stated that CD4+ T cells from RA patients are more responsive in T cell signal pathway.Then we identified an important mi RNA which could regulate T cell activation by its target m RNA.Finally we discussed the role of mi R-181 a in the regulation of CD4+ T cell activation in the pathogenesis of RA in vitro.These findings suggest the molecular mechanisms of T cell activation in Rheumatoid Arthritis and provide new ideas for the treatment of Rheumatoid Arthritis.Part Ⅰ The function study of CD4+ T cells and its subsets in Rheumatoid Arthritis Objective: To investigate the function of CD4+ T cell and its subsets activation in RAMethod:1.PBMC were isolated from 33 RA and 28 healthy controls by the Ficoll-Hypaque gradients.CD4+ T cells were further purified by the negative selection with magnetic beads using a CD4+ T cell isolation kit.2.Flow Cytometry analysed the expression of the calcium influx.3.The expression of activation marker CD25 and CD69 were detected by surface staining.4.The phosphorylation of LCK,ZAP70 and ERK were measured by BD Phosflow.Results:1.The CD4+ T cells and na?ve CD4+ T cells from RA patients displayed a significantly higher relative Ca2+ influx than that of healthy donors,P<0.001,P<0.01,respectively.There is no statistical difference in memory CD4+ T cells.A considerable positive correlation between Ca2+ influx and DAS28 was discerned,but there is no correlation between Ca2+ influx and age.2.The CD4+ T cells and its subsets from RA patients expressed apparently elevated CD25 and CD69 after CD3/CD28 microbeads incentive towards healthy controls.3.The CD4+ T cells and na?ve CD4+ T cells from RA patients after CD3/CD28 cross-linking stimulation were higher in parallel with healthy donors among ERK,ZAP70,LCK phosphorylation.In contrast,no similar discrepancy was seen in CD4+ memory T cells except LCK phosphorylation.Conclusion: The CD4+ T cells exhibited higher intracellular calcium influx,which was associated with disease activity.And the TCR pathway is over activated in CD4+ T cells,like overexpression of CD25 and CD69 and elevated LCK,ZAP70 and ERK phosphorylation.Meantime na?ve CD4+ T cells have strong calcium signals and increased ZAP70 and ERK phosphorylation,which there were similar expression in memory CD4+ T cells.So we focused on the na?ve CD4+ T cells as a study target in the following experiments.Part Ⅱ The establishment of mi R-181 a expression and its downstream regulation in na?ve CD4+ T cells from RAObjective: To analyze the mi RNA expression that can regulate na?ve CD4+ T cells over-activation in RA and establish the regulation of mi R-181 a and DUSP6.Method:1.PBMC were isolated from 18 RA and 18 healthy controls by the Ficoll-Hypaque gradients.The na?ve CD4+ T cells were further purified by the negative selection with magnetic beads using a na?ve CD4+ T cells isolation kit.2.Total RNA and protein were extracted from na?ve CD4+ T cells.3.Real-time PCR was used to determine the expression of mi R-181 a,DUSP6 and PTPN22.4.The protein level of DUSP6 was examined by Western Blot.5.The na?ve CD4+ T cells of RA was transfected with the mi R-181 a mimics or inhibitor and its respective controls.After 48 h,cells were harvested and assayed for m RNA and protein expression of DUSP6 by Real-time PCR and Western Blot.6.Luciferase assay was used to verify that DUSP6 is the target gene of mi R-181 a.Results:1.According to the Targetscan database,we initially consider that mi R-181 a is well suited for the study of regulated DUSP6 and PTPN22 expression,and then controls the T cell activation pathway.2.The levels of mi R-181 a in na?ve CD4+ T cells of RA were notably escalated in the RA patients compared with the healthy donors,P<0.001.DAS28 correlated positively with mi R-181 a expression,r=0.59,P<0.01.3.The m RNA expression of DUSP6 was lower in na?ve CD4+ T cells from RA patients than that from the healthy donors,P<0.05,while no difference of PTPN22 was monitored between two groups.Additionally,RA patients exhibited distinctly declined the protein expression of DUSP6 in na?ve CD4+ T cells compared to healthy donors,P<0.05.4.The DUSP6 m RNA levels decreased in na?ve CD4+ T cells of RA transfected with mi R-181 a mimic,but the protein expression was greatly lower,P<0.001.The na?ve CD4+ T cells transfected with mi R-181 a inhibitor greatly multiplied the m RNA and protein expression of DUSP6 in RA patients,but not those with mi R-181 a negative control,P<0.01.5.The 3’UTRs of DUSP6 was cloned into the psi Check2.2 luciferase expression vector in na?ve CD4+ T cells of RA patients and we examined luciferase activity upon mi R-181 a mimic.Ectopic expression of mi R-181 a significantly suppressed the luciferase activity of wild-type DUSP6 3’ UTR,but not its mutant 3’ UTR,revealing that mi R-181 a silenced DUSP6 expression through direct binding to their 3’ UTR.Conclusion: The highly expression of mi R-181 a promoted the occurrence and development of RA because mi R-181 a inhibited DUSP6 expression,which leaded to is the excessive activation of TCR pathway.The mi R-181 a / DUSP6 nexus plays an important role in the pathogenesis of Rheumatoid Arthritis.Part Ⅲ The role of mi R-181 a / DUSP6 nexus in na?ve CD4+ T cells of RAObjective: To probe the effect of mi R-181 a / DUSP6 nexus on the function of na?ve CD4+ T cells of RA.Method:1.PBMC were isolated from 12 active RA and 12 healthy controls by the Ficoll-Hypaque gradients.The na?ve CD4+ T cells were further purified by the negative selection with magnetic beads using a na?ve CD4+ T cells isolation kit.2.The na?ve CD4+ T cells of RA was transfected with the mi R-181 a inhibitor and its respective controls.3.Real-time PCR was used to determine the expression of mi R-181 a to verify the efficiency of transfection.4.Flow Cytometry analysed calcium influx.5.IL1-α,IL-2,IL6,IL-10,IFN-γ and TNF-α were detected by intracellular staining.6.Flow Cytometry was used to detect the differentiation of na?ve CD4+ T cells in RA.Results:1.The na?ve CD4+ T cells of RA transfected with mi R-181 a inhibitor and negative control.After 48 hours,total RNA was extracted and detected by RT-PCR to evaluate the transfection efficiency.The results showed that mi R-181 a expression decreased,which could be used for the following experiments.2.The relative calcium influx was lower along with the na?ve CD4+ T cells of RA transfected with mi R-181 a inhibitor than negative control.3.Compared with healthy donors,IL1-α,IL6,TNF-α and IFN-γ were up-regulated in RA,P<0.05,P<0.01,P<0.01,P<0.05,respectively;but IL-2 and IL-10 expressed similarly.Compared with the transfection of mi R-181 a negative inhibitor,down-regulation of mi R-181 a expression can significantly reduce the expression of IL1-α,IL6 and TNF-α,P<0.01,P<0.05,P<0.01,respectively.4.The na?ve CD4+ T cells of RA transfected with mi R-181 a inhibitor was against the differentiation into Th1 cells while favored the induction of a Th2 response compared to the negative control.Conclusions: In the na?ve CD4+ T cells of RA patients,inhibition of mi R-181 a expression by up-regulation of DUSP6 expression,thereby suppresses the ERK signaling pathway,reduces calcium influx,affects the activation of T cells,decreases the secretion of pro-inflammatory factors and inhibits autoimmune response.These findings shed light on the molecular mechanisms of T cell activation in RA and provide new ideas for the treatment of rheumatoid arthritis. |
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