The Mechanisms Through Which MiR-21-Tipe2 Regulates The Oral Tolerance And MiR-21-Pdcd4 Regulates The Glucose Tolerance

miR-21 is one of the early discovered human microRNAs（miRNAs）.It acts as an oncogene and plays an important role in cell differentiation,proliferation and apoptosis.Although great progress has been made on the role and the mechanism of miR-21 in tumorigenesis and development,more and more studies have found that miR-21 also play important roles in other biological functions,because miR-21 can regulate the expression of multiple targets.Dendritic cells（DC）play an important role in immune regulation.After differentiation,bone marrow-derived dendritic cells（BMDCs）are in a semi-mature state,and can promote immune tolerance.After stimulation,BMDCs become fully matured and are capable of activating immune response.The differentiation and development of DCs go through a process from semi-mature to fully mature,and DCs at these two stages can exert opposite regulatory effects on immune response.Previous studies have reported that miR-21inhibits the full maturation of DC in a renal ischemia-reperfusion model.Our study found that miR-21 inhibits dendritic cell semi-maturation during BMDCs differentiation.As a direct targets of miR-21,although tumor necrosis factor-α-induced protein 8-like 2（TNFAIP8L2 or Tipe2）inhibits stimulation-induced DCs fully maturation,Tipe2 promotes dendritic cell semi-maturation during the differentiation of BMDCs.Although the role and mechanism of miR-21 and Tipe2 in regulating the full maturation of DC have been well studied,the mechanisms by which miR-21 and Tipe2 regulate the semi-maturation of BMDCs and their role in immune tolerance are unclear.On the other hand,it has been reported that miR-21 expression is significantly up-regulated in the islets of patients with impaired glucose tolerance and study using isletβ-cell line revealed that miR-21 over-exoression suppresses GSIS（Glucose Stimulated Insulin Secretion）.However,considering the limitations of using cell lines and microRNAs over-expression,conclusions drawn from those studies still need to be verified in primary cells,preferably using knockout mice.In this study,we explored the mechanism of miR-21 and its target genes in regulating oral tolerance and glucose tolerance using miR-21-deficient mice(miR-21^-/-),Tipe2-deficient mice(Tipe2^-/-)and isletβ-cell specific deletion of miR-21 mice（miR-21βKO）,respectively.Major results of the current study were summarized as follows:1.miR-21-Tipe2 pathway inhibits dendritic cell semi-maturation and promotes oral toleranceWe found a negative correlation between the expression of miR-21 and Tipe2during the differentiation of BMDCs.miR-21 expression level is low in the early stage of BMDCs differentiationa,accompanied by relatively high expression of Tipe2.However,while the expression level of miR-21 was gradually increased in the late stage of BMDCs differentiation,the expression of Tipe2 was gradually decreased.Since Tipe2 is a known direct target of miR-21,we found that the expression of Tipe2is significantly higher in miR-21-deficient BMDCs as expected.Because the expression of maturition markers is increased in miR-21-deleted BMDCs but decreased in BMDCs lacking Tipe2,it is very likely that miR-21 inhibits DC semi-maturation through negative regulation of Tipe2 during the differentiation of BMDCs.Mechanistic study revealed that Tipe2 promotes BMDCs semi-maturation through the activation of PDK1-PKCδ-MAPK signaling pathway.Since semi-matured dendritic cells play important roles during the induction of peripheral regulatory T cells（pTregs）,in consistentency with these results,we found that deletion of Tipe2promotes the induction of pTregs in the intestinal mucosa.2.miR-21-Pdcd4 pathway maintains glucose toleranceWe found that mice with isletβ-cell specific deletion of miR-21（miR-21βKO）showed defects in maintaining glucose tolerance and insulin secretion.However,these results are different from the study using isletβ-cell line.Mechanistic studies showed that,althoughβ-cell specific deletion of miR-21 does not affect the number and size of isletβ-cell,insulin synthesis,and insulin transport and release,the expression of glucose transporter 2（Glut2）in the islets of miR-21βKO mice is significantly reduced.Further studies revealed that miR-21 promotes Glut2 expression via miR-21-Pdcd4-AP-1 signaling pathway.More importantly,our study found that increasing miR-21 level in the pancreas of type 2 diabetic db/db mouse significantly reduced blood glucose level.In conclusion,our study revealed that:1)during the differentiation of BMDCs,while miR-21 may inhibit DC semi-maturation through negative regulation of Tipe2expression,Tipe2 promotes the semi-maturation of BMDCs and suppresses oral immune tolerance by activating PDK1-PKCδ-MAPK signaling pathway;2)in isletβcells,miR-21-Pdcd4 signaling pathway maintains glucose tolerance through the promotion of Glut2 expression and hence insulin secretion.Increasing miR-21 level in the pancreas of type 2 diabetic mouse significantly reduced blood glucose level.Taken together,the current study suggests that miR-21 may be an important target for the prevention and treatment of inflammation-related diseases and type 2diabetes.