A Research Of The Role Of MiR-449a In The Activation And Metabolism Of CD4~+ T Cells

Recently,acute rejection after organ transplantation remains a risk factor for graft survival.The CD4~+ T cell-mediated immune response plays an important role in acute transplant rejection.Energy metabolism is important for the activation and function of CD4~+ T cell,because oxidative phosphorylation(OXPHOS)is required for the activation of T cells,while aerobic glycolysis is required for interferon cytokine production in T cells.MicroRNAs(MiRNAs)are small,non-coding single-stranded endogenous RNAs which are~22 nucleotides in length,and modulate gene expression through canonical base pairing to complementary sequences in the 3’UTR of target mRNA.Mi RNAs have been found to take part in the regulation of immune cells metabolism.However,the mechanism about how specific miRNA regulates the gene expression that is involved in cellular energy metabolism and then influences CD4~+ T cell function in the allotransplantation is still unclear.Further,the microRNA miR-449a increases during the acute rejection after allotransplantation,suggesting that miR-449a plays a relevant role in acute rejection.Mi R-449a is involved in the metabolism of fat and cholesterol.Besides,Notch is the target gene of miR-449a,and it can regulate the activation of immune cells by changing cellular mitochondrial respiration and OXPHOS.Therefore,it is promising to target at mi R-449a and observe its role in the acute rejection after allotransplantation,and the activation and energy metabolism of CD4~+ T cells.We examined the miR-449a expression in peripheral blood mononuclear cells(PBMCs)and graft infiltrating lymphocytes(GILs)between syngeneic transplant and allogeneic transplant groups on day 7 post-heart allotransplantation.We found that miR-449a expression in PBMCs and GILs significantly increased in the allogeneic heart transplant group compared to the syngeneic transplant group(P<0.01).It indicates that miR-449a has a connection with alloimmune response.Furthermore,we also examined the miR-449a expression in CD4~+ T cell activation,mixed lymphocyte reactions(MLRs)in vitro,and confirmed that the expression of miR-449a was significantly elevated in activated CD4~+ T cells.These results suggest that miR-449a may relate to the activation of CD4~+ T cells.To evaluate the effect of miR-449a on CD4~+ T cell energy metabolism,we inhibited the expression of miR-449a in CD4~+ T cells in vitro and then analyzed key metabolic parameters using XFp extracellular flux analyses including phenotype test,mito stress test and glycolysis stress test.We found that inhibition of miR-449a expression in this same CD4~+ T cell population resulted in decreased basal respiration and spare respiration capacity(SRC)in its mitochondrial respiration.It suggests that miR-449a influences the basal mitochondrial respiration and SRC of CD4~+ T cells.In summary,miR-449a plays an important role in the activation and energy metabolism of CD4~+ T cells.This is the first time the role of miR-449a in the activation and energy metabolism of CD4~+ T cells has been reported,and it provides a base for further researches.In addition,we offer new ideas and research basis to other researches about immune rejection and immune tolerance,which target at specific miRNA.