The Effect And Mechanism Of MiR-34a On Treg/Th17 Balance In CIA Mouse Model

Objective:Preliminary studies in our lab have demonstrated that IL-6 and TNF-? can disturb Treg/Th17 balance via NF-?B-mi R-34 a axis.In this study,EL-4 cells were transfected with agomi R-34 a and antagomi R-34 a in vitro.NF-?B inhibitor was used to block the NF-?B signaling pathway,which was intended to investigate the relationship of NF-?B,mi R-34 a and Foxp3.To explore the regulation of mi R-34 a on Treg/Th17,we use a collagen-induced arthritis(CIA)model by injecting agomi R-34 a and antagomi R-34 a.We also established a CIA model of mi R-34 a transgenic mice,the environmental factors were excluded compared with DBA model.Taking the Treg/Th17 balance as an entry point to illuminate the mechanism of mi R-34 a on CIA.This will be useful for the treatment of autoimmune diseases.Methods:(1)Mouse lymphoma cell line cells(EL-4)were transfected with agomi R-34 a for36h and then treated with TGF-? for 12 h.The group of EL-4 cells transfected with antagomi R-34 a was treated with TGF-?,IL-6,TNF-? for 12 h to identify Foxp3 level by Immunoblot.(2)Mouse Na?ve CD4+T cells purified by MACS from spleen were cultured with TGF-? to induce Treg in vitro.Transcriptional levels of mi R-34 a in different groups,which were treated with IL-6,TNF-? and NF-?B inhibitor respectively for 24 h,were detected by q PCR.Foxp3 protein levels were detected by Immunoblot.(3)Establishing CIA model in DBA1/J mice.The agomi R-34 a and antagomi R-34 a were injected into CIA mice and observed the role of mi R-34 a in the development of the disease(clinical severity score,imaging test,pathology).The proportion of Treg/Th17 in spleen and lymph nodes CD4+T cells was detected via flow cytometry.(4)Establishing CIA model in mi R-34 a transgenic mice.The susceptibility of CIA induction was compared between the transgenic mice and wild type mice.The assessment methods were introduced as above.Results:(1)Foxp3 protein level is reduced in EL-4 cells which were transfected with agomi R-34 a while antagomi R-34 a can upregulated Foxp3 protein level in EL-4cells.(2)IL-6/TNF-? can upregulate the expression of mi R-34 a and decrease the level of Foxp3.NF-?B inhibitor can reverse the above results.(3)The model of CIA in DBA mice was established successfully.agmi R-34 a can increase the severity of arthritis,and the opposite results were observed in antagomi R-34 a group.The peak score of CIA-agomi R-34 a group is 11 and the average score of CIA-antagomi R-34 a is 3-4.Histopathological analysis and imaging test showed that antagomi R-34 a was associated with a marked reduction in inflammation.FCM showed that the proportion of Treg cells in agomi R-34 a group was decreased,while Th17 cells were increased in spleen and lymph nodes.In addition,antagomi R-34 a can effectively reverse the imbalance of Treg/Th17.(4)The model of CIA in mi R-34 a transgenic mice was established successfully.The results of clinical severity score,histopathological analysis and imaging test shoewed that mi R-34 a can increase the condition of CIA.Conclusion:Our results further demonstrated that the IL-6/TNF-?-NF-?B-mi R-34 a axis can disturb Treg/Th17 balance.mi R-34 a can upregulate Th17 cells and downregulate Treg cells in spleen and lymph nodes.While antagomi R-34 a can effectively recover Treg/Th17 balance,which is accordance with the severity of CIA.