The Role Of MicroRNA-451a In Immunologic Derangement In Patients With Dilated Cardiomyopathy

Part I:The relationship between abnormal activation of CD4+T cells and miRNAs in patients with dilated cardiomyopathyObjective:Previous studies have revealed that the abnormal activation of CD4+T cells in patients with dilated cardiomyopathy(DCM)might be associated with the immunopathogenesis of the disease.However,the underlying mechanisms remain largely undefined.Our aim was to investigate the relationship between abnormal activation of CD4+ T cells and miRNAs in patients with DCM.Methods:We recruited 50 patients with DCM and 50 age-and sex-matched controls.The peripheral blood mononuclear cells(PBMCs)were isolated from DCM patients and controls.The expressions of activation marker CD25,CD69 and MHC-Ⅱ of CD4+T cells were detected by flow cytometry.The proliferation of CD4+T cells in response to anti-CD3/28 was calculated by measuring the dilution of CFSE.CD4+T cells were purified from PBMCs by magnetic activated cell sorting(MACS)using a human CD4+T Cell Isolation Kit.A total RNA of CD4+T cells was extracted and was used for the expression analysis with miRNA 4.1 arrays and bioinformatics analysis.Results:(1)Compared with the control group,CD4+T cells from DCM patients showed increased expression levels of CD25 and CD69 and enhanced proliferation in response to anti-CD3/28,indicating an activated state,whereas the expression of the late activation marker MHC-Ⅱ on CD4+T cells showed a non-significant trend towards an increase in the DCM patients.(2)The purity of magnetically sorted CD4+T cells was>97%.(3)The miRNA expression profiles of CD4+T cells in 5 DCM patients and 5 age-and sex-matched controls were screened.We obtained a list of 17 miRNAs that were significantly over-or under-expressed with more than 2-fold changes in the patients with DCM to controls.Among them,15(miR-206,miR-595,miR-297,miR-1180-3p,miR-3148,miR-4701-3p,miR-3064-5p,miR-4793-3p,miR-6785-5p,miR-6796-5p,miR-6807-5p,miR-6847-5p,miR-6849-5p,miR-6856-5p,and miR-7844-5p)were up-regulated and 2(miR-451a and miR-486-3p)were down-regulated in the patients with DCM.(4)The down-regulation of miR-451a was further validated in purified CD4+ T cells from an additional 45 DCM patients and 45 controls by RT-PCR,which was consistent with the result of miRNA chip.To adjust the potential confounding effect on miR-451a expression for the variables factors,we performed a multiple linear regression analysis.The results indicated that diabetes,or medical therapies did not influence miR-451a expression in CD4+T cells.Conclusions:Our results showed that the expression levels of surface immune activation markers CD25 and CD69 were significantly increased and the proliferation in response to anti-CD3/28 stimulation was enhanced in CD4+T cells of DCM patients,indicating that the CD4+T cells are abnormally activated in DCM patients.The miRNAs profile was altered in CD4+T cells from patients with DCM.Especially,the down-regulation of miR-451a may play an important role in abnormal activation of CD4+T cells in DCM.Part II:miR-451a regulates the activation and proliferation of T cells by targeting MycObjective:On the basis of Part I,to further explore whether miR-451a could regulate the activation and proliferation of T cells and to search its targeting gene.Methods:The Jurkat T cells were transfected with miR-451a mimics or inhibitors.The expression of surface activation markers CD25 and CD69 was measured by flow cytometric analysis.The level of IL-2 in cell supernatant and proliferation ability of cells were detected by ELISA and CCK-8,respectively.Bioinformatics tool predicted that Myc may be the target gene for miR-451a.The binding ability of miR-451a to Myc 3’-UTR was detected by luciferase reporter assay.The Myc expression level was measured in Jurkat T cell transfected with miR-451a mimics or inhibitors.The Jurkat T cells were transfected with siRNA mediated down-regulation of Myc.The Myc expression levels were measured by RT-PCR or western blot,and the activation and proliferation of Jurkat T cells were detected.The expression of Myc mRNA levels in CD4+T cells was analyzed by RT-PCR,and the correlation between miR-451a and Myc was calculated by two-sided Spearman’s test.Results:(1)Compared with negative controls(NC),the miR-451a mimic inhibited the expressions of surface activation markers CD25 and CD69 in Jurkat T cells,and down-regulated the concentrations of IL-2 in cell supernatant and proliferation ability of cells,whereas the miR-451a inhibitor produced the opposite effects.(2)The binding ability of miR-451a to Myc 3’-UTR was validated by a dual-luciferase reporter assay.The miR-451a mimic inhibited the expression of Myc in mRNA and protein levels,whereas the miR-451a inhibitor produced the opposite effects.(3)Compared with siRNA NC group,the expression of Myc in mRNA and protein levels were inhibited in Jurkat T cells transfected with siRNA-Myc.siRNA-Myc down-regulated the expressions of surface activation markers CD25 and CD69,and decreased the production of IL-2 in cell supernatant and proliferation ability.(4)The expression of Myc was significantly up-regulated in CD4+T cells from patients with DCM.A strong inverse correlationship was observed between the Myc and miR-451a in CD4+T cells from patients with DCM.Conclusions:miR-451a regulates the activation and proliferation of CD4+T cells by targeting Myc in patients with DCM,and the down-regulation of miR-451a may be an important mechanism for abnormal immune activation in DCM...