Role Of MiR-568in The Activation Of Human CD4~+t Lymphocyte And The Mechanism Study

MicroRNAs (miRNAs) are a class of small single-stranded RNA, the length of about18-22nucleotides (nt). It is generated by DNA transcription, but not translated intoproteins, but regulate the function of other genes in protein synthesis. Therefore, miRNAis a single-stranded RNA which reversed complementary for the mRNA of otherprotein-coding genes, is a gene regulator of other protein-coding genes. lin-4and let-7arefirst microRNAs that were observed in C.elegans, the development of the C.elegans wascontrolled by these miRNA. Subsequently, miRNA were found in from C.elegans toDrosophila and human multiple tissues, suggesting that these molecules may represent agene family evolved from the ancient ancestors of the sRNA, and retain a high degree ofevolutionary conservation between various species. MicroRNA is an important regulatorymolecule to regulate the expression of other genes, it participates in the importantprocesses of life course, including developmental processes, hematopoietic process, tumordevelopment and differentiation, and development and metabolism of many importantbiological processes.The adaptive immune is the defense mechanisms of a high specificity degree againstof the antigen-specific substances in the immune system during evolution, including theT-cell-based cellular immunity and B-cell-besed humoral immune. The mammalianimmune system plays an important role in anti-infection, homeostasis and immunesurveillance. In order to achieve the right balance between immune response and immunetolerance, the initial immune response and termination and intensity of reaction must be rigorous adjusted. Relative to the regulation of transcription factors fully on or off,miRNAs are more suitable for immune system with fine-tuning and quantitative regulation,because they are regulation by a very short sites of action, and in the sites of action, one ora few bases can play a key role, and because they are easy to have mutations in theevolutionary process, which also play a role in gene regulation with the pathogenevolution repository organisms. Recent studies indicate that miRNAs indeed play animportant role in the fine regulation of the immune system. In-depth understand of themechanism how miRNAs regulate the immune system not only can help us to identify anew target for the immune response, but also can help build effective miRNA therapy.Despite the research that detect the change of microRNA expression in na ve CD4~+Tcells、effecyive CD4~+T cells and memory CD4~+T-cell, but the real role of thedifferentially expressed miRNA in T cell activation process remains unclear. Therefore,further study of these miRNA in T cell activation and differentiation will help us to bettercontrol the adaptive immune response.Up to now, we don’t see the reports of miR-568function and immune-relatedfunction. If miR-568, which devreased during the activation of Jurkat cells may play a rolein the regulation of CD4~+T lymphocyte activation? What is the target of miR-568in theactivation of CD4~+T lymphocyte? These questions will be discussed.Objective：1) To select T cell activation-related microRNAs;2) To study The role of miR-568during the activation of CD4~+T lymphocyte;3) Explore the downstream target gene of miR-568during the activation of CD4~+Tlymphocyte;4) To study the function of miR-568in the differentiation and function of Treg cells..Methods：1) Selected T cell activation-related microRNAs by miRNA array； 2) Detected the expression of miR-202*、miR-33b、miR-568and miR-576usingqRT-PCR in Jurkat and human CD4~+T lymphocytes before or after activation;3) Constructed hsa-miR-568eukaryotic expression vector, synthetic miR-568mimics;4) Transfected the miR-568into Jurkat and human CD4~+T lymphocyte beforestimulation, and then detected the surface activation marker CD25、CD69and CD154byflow cytometry;5) Predicted the target genes of miR-568by Bioinformatics prediction methods;6) Constructed the dual-luciferase reporter gene vector, validate the target gene ofmiR-568by luciferase reporter assay、Real time RT-PCR and Western Blot;7) Transfected miR-568into Jurkat and human CD4~+T lymphocyte before stimulation,and then detected the expression of IL-2by Real time RT-PCR、ELISA, and detected theproliferation of human CD4~+T lymphocyte by MTT;8) Isolated human CD4~+CD25~+Treg cell by using immunomagnetic beads, and detectedthe expression of miR-568by qRT-PCR after activation;9) Detected the level of IL-10and TGF-β in CD4~+CD25~+Treg cell s by ELISA aftermiR-568transfection;10) Detected the proliferative capacity of Treg cells by MTT assay;11) Detected the effection of miR-568on Treg cells to suppress the proliferative capacityof effector T cell by CCK-8assay;.12) Knockdown NFAT5by siRNA in CD4~+CD25~+Treg cells, detected IL-10, TGF-βsecretion levels by ELISA;13) Knockdown NFAT5by siRNA in na ve T cells and detected the differentiation ofna ve T to Treg cells.Results： 1) hsa-miR-568significantly inhibite the activation of Jurkat and human CD4~+Tlymphocyte.We found that four microRNAs were significantly downregulated in activated Jurkat cells(miR-202*、miR-33b、miR-568and miR-576) by use miRNA array. The experiments ofqRT-PCR show that expression of thes four microRNAs are significantly decreased in theactivated Jurkat and human CD4~+T cell. Here, we focused on hsa-miR-568molecule, andwe detected the expression of hsa-miR-568at different time points and found that theexpression levels of hsa-miR-568were significantly reduced in Jurkat and human CD4~+Tlymphocyte cells at12h,24h and72h after activtion. Flow cytometry results demonstratethat transfection of miR-568could significantly inhibit Jurkat and human CD4~+Tlymphocyte activation level.2) NFAT5is the direct target gene of miR-568is NFAT5, miR-568may affect theexpression level of IL-2to inhibit the activation of CD4~+T lymphocytes viaNFAT5. This function is validated by siRNA mediated NFAT5knockdown.We further to screaned the target genes of miR-568by target gene prediction software.TargetScan and miRanda were used to predict target genes of miR-568, results show thatfive candidate target genes are associated with T-cell activation: NFAT5、CD28、CD3G、CD96and CD69. Real-time quantitative PCR and Western blot results confirmed thatmiR-568can inhitit the protein expression level of NFAT5, but did not affect mRNAexpression levels. The dual luciferase reporter gene experiments results confirmed thatmiR-568can bind with the3’UTR of NFAT5. Result of ELISA proved that miR-568candownregulate expression levels of IL-2. MTT assay showed that miR-568can inhibit theproliferation of CD4~+T lymphocyte.3) In Treg cell, miR-568can inhibite Treg cell activation、 proliferation anddifferentiation, inhibit the secretion of IL-10and TGF-β, and inhibit itsimmunosuppressive funtion.CD4~+T lymphocytes can differentiate into CD4~+CD25~+Foxp3+Treg, while the NFAT family are closely related to Treg cell, and our experiments proved that, miR-568affectedthe biological function of Treg cells such as activation, proliferation, differentiation,upsokine secretion and immune suppression function.Conclusion：miR-568is a CD4~+T lymphocyte activation-related miRNA, its downstreamtarget gene is NFAT5, miR-568inhibit expression level of IL-2through NFAT5toregulated the CD4~+T lymphocytes activation; miR-568significantly inhibited thedifferentiation and function of regulatory CD4~+T cell subset (Treg).