Circular RNA MAP2K2 Regulating Dendritic Cells-mediated Alloimmune Rejection In Heart Transplantation

BACKGROUND:As a worldwide common disease,cardiovascular disease poses a great threat to human lives safety and quality.Nowadays,the material well-being living standards has also increased the number of cardiovascular disease patients in China year by year.For patients with cardiovascular disease that progress to the end stage,medical treatment often can’t significantly relieve the pain caused by severe heart failure.For these patients,heart transplantation is their last chance to get recovery.It has been over a half century after the completion of the first heart transplantation surgery.With the improvement of medical technology,the postoperative survival status of heart transplantation patients has increased from 18 days to 80%-90%first-year survival rate,which proofed that the heart transplantation surgeries and postoperative management programs have been continuously improved.However,the long-term survival rate of patients(10-15 years)has not been greatly improved.Except for the impact of chronic immune rejection,the non-specific immunosuppression caused by the current use of immunosuppressant therapy is also one of the important factors.Many patients will lose their lives due to complications such as infection or cancer.Therefore,how to produce specific immunosuppression of grafts has become one of the most important issues in the field of transplantation.So we focus on the immune system,more specifically,the dendritic cells in the immune system.Dendritic cells,named after their dendritic pseudopodia on their surface,are the most powerful antigen-presenting immune cells found so far.Mature dendritic cells can participate in the activation of T cells by presenting antigens.They can also secret cytokines and chemokines,thereby participating in the innate and adaptive immune processes.Immature dendritic cells do not have antigen presentation ability,on the contrary,they have stronger migration and antigen phagocytosis ability.For these reasons,the dendritic cell also plays a very important role in the immune rejection of heart transplantation.Studies have shown that donor DCs in transplanted hearts were rapidly replaced by recipient DCs,mediating transplant rejection by stimulating effector T cells.Clearance of receptor DCs can eliminate immune rejection which represents the great potential of dendritic cells to be the target of inhibiting immune rejection of heart transplantation.Dendritic cells can transform into a special type of mature dendritic cells after being stimulated by special human factors,as called as immune-tolerant dendritic cells(Tol-DCs).Such dendritic cells can phagocytose antigens and present antigens,but the function of immune cells stimulated by TolDCs is inhibited.The modified Tol-DCs have been shown to significantly prolong the survival time of transplanted hearts in mice.The formation of Tol-DCs can already be induced by applying cytokines such as IL-10,IL-27.but the induction of Tol-DCs and how to use them as a tool for immunotherapy still need further investigation.Circular RNA is a kind of closed-loop RNA formed by a special splicing method.Because the junction sites are covalently linked,they tend to have better stability than their linear transcripts.Even circular RNA was initially thought to be a biologically inactive production,with the deeper investigation,it was found to be not only abundant in eukaryotic cells,but also highly expressed with highly tissue-specific,which means that circular RNAs are more than just useless metabolites.At present,many studies have shown that circular RNA was closely related to a variety of physiological and pathological processes.In immunization and organ transplantation,circular RNA also showed great potential.Our previous experimental results show that the expression of circMAP2K2,a circular RNA,which home gene is MAP2K2,differs significantly between mature and immature dendritic cells.In this study,it was expected that by adjusting the expression of circMAP2K2 in dendritic cells we can induce the formation of immunosuppressive dendritic cells.Thereby the survival time of mouse transplanted heart can be prolonged.We also want to explore the molecular mechanism behind it.Research Objectives:1.To clarify the expression level and location of circMAP2K2 in dendritic cells.2.To explore the impact of changes in circMAP2K2 expression level on the phenotype of dendritic cells.3.To explore the impact of changes in circMAP2K2 expression level on the immune function of dendritic cells.4.To explore the possible molecular mechanisms underlying the regulation of dendritic cell phenotype and immune function by circMAP2K2.5.To establish a mouse allogeneic heart transplantation model and explore whether dendritic cells induced in vitro to downregulate circMAP2K2 expression have immunosuppressive function.6.To verify whether the immunosuppression caused by dendritic cells downregulating circMAP2K2 expression is antigen-specific.METHODS AND MATERIALS:1.Cultivating dendritic cells in vitro and detecting the expression level and location of circMAP2K2.(1)Bone marrow cells are extracted from mice.These cells are then induced differentiation into dendritic cells using cytokines in vitro,and are retained at different time points during the culture process for flow cytometry to detect maturity related surface molecule expression levels.Simultaneously retain RNA samples and use qRT PCR to detect the expression level of circMAP2K2 at different time points.(2)RNA in situ hybridization was performed on cultured mature dendritic cells,and biotinylated probes targeting the circMAP2K2 junction were co incubated with dendritic cells.The expression position of circMAP2K2 in dendritic cells was determined using fluorescence microscopy.2.Using siRNA to downregulate the expression of circMAP2K2 and observing the effects of downregulation of circMAP2K2 expression on dendritic cell phenotype and immune function.(1)siRNA targeting circMAP2K2 is transfected into dendritic cells cultured in vitro using liposomes.After 48 hours of incubation,transfected cell RNA is extracted and using qRT PCR technology to detect the expression levels of circMAP2K2 and MAP2K2 mRNA.Using Western Blot to detect the expression levels of MAPK signaling pathway related proteins to confirm that siRNA only regulates the expression level of circMAP2K2.(2)siRNA targeting circMAP2K2 is transfected into dendritic cells cultured in vitro using liposomes.After 48 hours of incubation,flow cytometry analysis is performed on the cells to observe the expression levels of surface maturation related molecules to confirm the phenotypic changes of dendritic cells.(3)siRNA targeting circMAP2K2 is transfected into dendritic cells cultured in vitro using liposomes,extract total RNA after 48 hours of incubation.Then we detect the mRNA expression level of pro-inflammatory cytokines to determine the immune function changes of dendritic cells.(4)siRNA targeting circMAP2K2 is transfected into dendritic cells cultured in vitro using liposomes.After 48 hours incubation,we mix transfected cells with spleen cells of different strains of mice in proportion for mixed lymphocyte culture.After co culturing for a period of time,the proliferation of T cells and the differentiation of Treg cells from different strains of mice were detected to determine the changes in immune system function caused by downregulating circMAP2K2 dendritic cells.3.Detecting proteins that may be related to circMAP2K2,exploring their expression changes then determining the molecular mechanisms behind the changes in dendritic cells by studying the expression levels of related signaling pathway proteins.(1)RNA immunoprecipitation(RIP)was performed on dendritic cell lysate,and qRT PCR was performed on the obtained samples to confirm the enrichment of circMAP2K2.Subsequently,mass spectrometry analysis was performed on the extracted material to identify potential related proteins.(2)Protein immunoprecipitation(Co IP)is performed on the target protein found,then we perform qRT PCR on the extract to confirm that the protein indeed interacts with circMAP2K2.(3)The total protein of dendritic cells downregulated by circMAP2K2 is extracted.Then we detect the expression levels of the target proteins mentioned above to determine the expression trend of the target proteins.Simultaneously,we detect the expression level and location of key proteins in potential related signaling pathways.4.Exploring the effects of downregulated circMAP2K2 dendritic cells on allogeneic heart transplant mice and investigating the characteristics of induced immunosuppression.(1)Preconditioning of recipient mice for allogeneic heart transplantation was performed by injecting dendritic cells with downregulated circMAP2K2 expression through the tail vein.After culturing for a period of time,heterotopic heart transplantation was performed.After short-term application of immunosuppressants,the immune rejection of heart transplantation was determined by touching the beating of the donor heart.(2)Pathological staining on the donor heart is performed to determine the degree of damage to the donor heart.(3)Spleen cells from recipient mice are extracted for flow cytometry detection.We observe the changes in the differentiation degree of Treg cells to determine whether the recipient mice have developed immune suppression.(4)The spleen cells of recipient mice are mixed with those of donor mice of the same strain or other strains of mice,and after a period of time,detect the proliferation of T cells in recipient mice to determine whether the immune suppression produced by recipient mice is antigen-specific.Results:1.The expression level of circMAP2K2 in dendritic cells increases with maturity.2.CircMAP2K2 is mainly expressed in the cytoplasm.3.siRNA targeting the circMAP2K2 junction site can downregulate the expression level of circMAP2K2 in dendritic cells.4.SiRNA targeting the circMAP2K2 junction site does not affect the expression of the ERK pathway in the MAPK signaling pathway.5.The decrease in the expression level of circMAP2K2 downregulated the expression of mature related surface co stimulatory molecules in dendritic cells.6.The decreased expression of circMAP2K2 downregulated the expression of pro-inflammatory cytokines and CCR7.7.The decreased expression of circMAP2K2 inhibited the pro proliferative effect of dendritic cells on T cells.8.The decreased expression of circMAP2K2 promoted the differentiation promoting effect of dendritic cells on Treg cells.9.CircMAP2K2 binds to SENP3 protein.10.The decrease in the expression level of circMAP2K2 inhibited the expression of SENP3 protein.11.Decreased expression of circMAP2K2 inhibits NF-κ The expression of p65 and p-p65 in the B signaling pathway.12.The decrease in the expression level of circMAP2K2 inhibited the process of p-p65 nuclear transfer.13.Dendritic cells that downregulated the expression of circMAP2K2 prolonged the survival time of donor hearts in allogeneic heart transplantation.14.Dendritic cells that downregulated the expression of circMAP2K2 alleviated inflammatory cell infiltration and fibrosis in the donor heart.15.Dendritic cells that downregulated circMAP2K2 expression induced Treg cell differentiation in heart transplant recipients.16.Dendritic cells that downregulated the expression of circMAP2K2 exhibit antigen-specific immunosuppression.