Screening And Function Identification Of MiRNAs Associated With Human CD4~+T Lymphocyte Activation

MicroRNAs are about 22-nucleotide long endogenous non-coding small RNAs that posttranscriptionally modulate gene expression by binding to the complementary sequence in the 3'untranslational region of target mRNA and initiate either their cleavage or a reduction in the translational efficiency. They were identified existing widely in C. elegans, Drosophila, Plant, Mammalian even virus and are highly conserved in different species. MiRNAs express tissue-specific and time-specific. They are key modulator to other functional genes and play an important role in various life activities of organism. miRNAs control a wide array of biological processes, including developmental process, hematopoiesis, organogenesis, proliferation, apoptosis, and tumorigenesis. Adaptive immunity response is the defense mechanism with the highly specificity generated in the early species evolution according to the specific antigen, including cellular immunity and humoral immunity. miRNAs have exerted huge effect in this complex system.Although it is reported that the different expression in naiveCD8+ T lymphocyte, effector CD8+ T lymphocyte and memory CD8+ T lymphocyte, the real function of the miRNA in the T lymphocyte activation is still unclear. The further studies on the differentially expressed miRNAs are needed to understand their roles in the activation or differentiation process. These studies may ultimately pave the way to manipulate the process of adaptive immunity.After testing the different expression between Jurkat cell and Jurkat cell activated with anti-CD3 antibody and anti-CD28 antibody by Exiqon miRNA array, five miRNAs were fond down-regulated in activated Jurkat cell. Quantitative real-time PCR confirmed the result. Because it has been reported that miR-181 family member, miR-181a, has close relation with the differentiation of T cell and B cell, we chose miR-181c as the target miRNA.Human CD4+ T cell were isolated from healthy human blood by beads and activated by anti-CD3 antibody and anti-CD28 antibody in order to verify the relationship between miR-181c and human CD4+ T cell. The result concluded by quantitative real-time PCR that the expression of miR-181c down-regulated in activated human CD4+ T cell is concord with the one in Jurkat cell.The target molecular of miR-181c was predicted on the web http://microrna.sanger.ac.uk/ and http://www.targetscan.org/. It was found that IL-2,CD69,IL1A,MAP3K3,MAP3K10 and NFAT5 that are associated with the signal way of T cell activation maybe the targets of the miR-181c and miR-181c can bind to the 3' untranslational regions of them. Especially, there are two binding sites in the 3' untranslational region of IL-2 and predicted score of IL-2 was the second of all the target protein. Dual-luciferase reporter assay shows miR-181c can bind to the 3' untranslational regions IL-2. So we mainly verified the relationship between miR-181c and IL-2 in the subsequent experiment.Jurkat cell was simulated as CD4+ T cell for his character. After was transferring with miR-181c, Jurkat cell was activated by double signals. CD25,CD69 andCD154 tested by flow cytometry shows that miR-181c can partly repress the activation of T cell.After cotranfected HEK293 cell with miR-181c and IL-2 3' UTR or IL-2 3' UTR mutant, it shows miR-181c repress the 50 percent of the expression of dual luciferase reporter containing the whole IL-2 3' UTR, and the expression of dual luciferase reporter only containing binding site2 is 33 percent whereas the expression of that only containing binding site1 is about 80 percent. It demonstrates the repression of the binding site2 is stronger.In order to further study whether IL-2 is the direct target of miR-181c or miR-181c down-regulated the expression of IL-2 by repressing the activation of T cell, the eukaryotic expression vector of IL-2 was constructed. The vector of pcDNA3-IL2CDS-UTR contains both the coding region and 3' untranslational region of IL-2, but the vector of pcDNA3-IL2CDS only contains the coding region of IL-2. When transferred with the two vectors, HEK293 cell which does not express IL-2 normally highly expresses IL-2. After cotransferring miR-181c and the vector to HEK293 cell and detecting the expressed level of IL-2 in the supernatant of the cell by ELISA, it was revealed that the expressed level of IL-2 down-regulated in the supernatant of the cell cotransferred with miR-181c and the vector pcDNA3-IL2CDS-UTR and expressed level was unchanged in the supernatant of the cell cotransferred with miR-181c and the vector pcDNA3-IL2CDS. It shows that miR-181c can repress the expression of IL-2 by binding to the 3' untranslational region and that IL-2 is the direct target of miR-181c.After transferred with miR-181c, human CD4+ T cell was activated by double signals. CD25,CD69 and CD154 tested by flow cytometry shows that miR-181c can partly repress the activation of human CD4+ T cell.The activated human CD4+ T cell transferred with miR-181c express lower level of IL-2. It is concluded that miR-181c can repress the synthesis and secretion of IL-2. It is observed that the mRNA of IL-2 was up-regulated in activated human CD4+ T cell, but it was not down-regulated after transferred with miR-181c. The same phenomenon was also observed in Jurkat cell, suggesting that miR-181c binds to the incomplete complementary sequence in the 3' untranslational region of IL2 and blocks the translation of IL-2 mRNA rather than degrades the mRNA of IL-2. It is in the accordance with the previous reported.Human CD4+ T cell was respectively transferred with miR-181c and N.C.. Seventy two hours after activated by anti-CD3 antibody and anti-CD28 antibody, the proliferation of human CD4+ T cell was detected by MTT assay. miR-181c reduces the activation of human CD4+ T cel due to the down-regulation of IL-2.All above results demonstrate that miR-181c has close contact with the activation of human CD4+ T cell. It can repress the activation and the expression of IL2 in human CD4+ T cell. It is verified by ectopic expression of IL-2 that miR-181c can directly repress the expression of IL-2 by binding to the 3'untranslational region of it. The one of the mechanisms of miR-181c represses the activation of human CD4+ T cell is that it down-regulates the expression of IL-2.It need further study on whether miR-181c indirectly inhibits the activation of human CD4+ T cell by other signaling moleculars that are the targets of it.