The Role Of MicroRNA Let-7 Regulates T Cell Function By Target Binding Nr4A1 In Heart Transplantation Rejection

BackgroundHeart transplantation is currently the most effective treatment for end-stage heart failure.The application of immunosuppressive agents allows most acute rejection to be effectively controlled,the use of immunosuppressive agents has led to the effective control of most acute rejection reactions,however,subsequent chronic rejection and immunosuppressant-related side effects,such as infection,malignancy and nephrotoxicity,have become major factors influencing the prognosis of patients.Nr4A1?Nuclear Receptor Subfamily 4 Group A Member 1?is a nuclear orphan receptor that plays an important role in T cells,however,the effect of Nr4A1 deficiency on T cell function and its role in cardiac allograft rejection,and the underlying regulatory mechanisms of Nr4A1 are unclear.ObjectiveA variety of gene knockout mice are used to establish heart transplant model to study the effect of Nr4A1 deficiency in T cells on heart transplantation rejection,clarify the regulatory mechanisms of Nr4A1 in T cells,find out upstream targets to modulate Nr4A1,and provide a new theoretical basis for the prevention and treatment of transplant rejection.Methods1.Observation of the changes of Nr4A1 expression in mouse heart transplantation modelUsing Balb/c mice as donors and C57BL/6 mice as recipients,a model of ectopic peritoneal heart transplantation was constructed,then qPCR and flow cytometry were applied to detect Nr4A1 expression in CD4⁺T cells from mixed lymphocyte culture in vitro.2.Exploration of the effect of Nr4A1 knockout on cardiac allograft rejection and CD4⁺T cell phenotypeBalb/c mice were used as donors,while Nr4A1^-/-mice or immune-reconstructed Rag1^-/-mice were used as recipients to establish heart transplantation models.The effect of Nr4A1knockout on cardiac allograft rejection and CD4⁺T cell phenotypic change were evaluated.3.Clarification of the effect of Nr4A1 agonists on CD4⁺T cell function and cardiac allograft rejectionHeart transplantation model was established with Balb/c mice as donors and C57BL/6 mice as recipients,then the effects of multiple specific small molecule agonists of Nr4A1 on cardiac allograft rejection were studied.Primary CD4⁺T cells were treated with agonist intervention and flow cytometry was used to detect T cell phenotypic changes.4.Investigation of the potential regulatory targets of Nr4A1 on a cellular levelTo study the effect of FK506 on the expression of Nr4A1 in CD4⁺T cells.Western Blot and qPCR were utilized to detect the regulation of Nr4A1 expression in CD4⁺T cells,and bioinformatics technology was applied to analyze the potential upstream targets of Nr4A1,then the effectiveness and specificity of upstream targets was validated by transfection and dual luciferase reporter assays.5.Construction of microRNA antagonists and observation of their effects on cardiac allograft rejectionThe target microRNA was identified as a potential upstream target of Nr4A1 based on the above experimental results,and a specific microRNA antagonist was constructed.The effects of the antagonist on regulation of Nr4A1 and cardiac allograft rejection were assessed,and the dependence of the protective effect of the specific microRNA antagonist on Nr4A1 was verified by Nr4A1^-/-mice.Results1.Nr4A1 expression was significantly up-regulated in recipient immune organs and CD4⁺T cells from lymphocyte mixed culture in vitro after heart transplantation;2.Nr4A1 knockout led to aggravating heart transplant rejection,and resistance to established induction programs of allograft tolerance;3.The experiments of cell adoptive reconstruction of mice immune system demonstrated that Nr4A1 mainly affected the apoptosis of activated CD4⁺T cells and the differentiation of Tregs cell subsets,thus influenced the rejection process;4.FK506 exerted post-transcriptional inhibition of Nr4A1 by up-regulating miRNA let-7a;5.MicroRNA let-7a antagonists could alleviate heart transplant rejection,and combined use of microRNA let-7a antagonists might reduce the dosage of FK506 and prolonged allograft survival.6.MicroRNA let-7a antagonists protected heart grafts by up-regulating Nr4A1 protein expression.ConclusionsNr4A1 plays an important role in CD4⁺T cell-mediated cardiac allograft rejection.Nr4A1deficiency can inhibit the apoptosis of activated CD4⁺T cells and the differentiation of Tregs cell subsets,aggravating the occurrence of rejection.Clinically used immunosuppressant FK506 inhibits the post-transcriptional translation of Nr4A1 by up-regulating microRNA let-7a.The specific antagonist of microRNA let-7a can reverse the inhibitory effect of FK506 on Nr4A1,thereby significantly prolonging graft survival and enhancing FK506 effect.Nr4A1 may be a new target for inducing graft immune tolerance after heart transplantation clinically.