【作者】 薛茜；

【导师】 杨安钢； 王涛；

【作者基本信息】 第四军医大学 ， 免疫学， 2009， 博士

【摘要】 微小RNA(microRNA,miRNA)是一类长度约为22 nt、内源性的小分子非编码RNA,通过和靶mRNA的3’末端非翻译区(3’ untranslational region,3’ UTR)完全互补或部分互补结合,使mRNA降解或抑制其翻译。研究发现,miRNA在线虫、果蝇、植物、哺乳动物、甚至病毒中广泛存在,在各个物种间具有高度的进化保守性。miRNA在表达上具有组织和时间的特异性,是调节其他功能基因表达的重要调节分子,在生物体的各种生命活动过程中发挥着重要作用,参与生命过程中一系列重要进程,包括发育进程、造血过程、器官形成、凋亡、细胞增殖和肿瘤的发生。适应性免疫是免疫系统在进化过程中形成的针对特定抗原物质的高度专一性的防御机制,包括以T细胞为主的细胞免疫和以B细胞为主的体液免疫。在这个复杂的系统中miRNA发挥着巨大的作用。尽管有研究探索了naiveCD8+ T细胞,效应性CD8+ T细胞和记忆性CD8+T细胞中miRNA的表达变化,但是在T细胞的活化过程中差异表达的miRNA的真正的作用仍然不清楚。所以进一步深入研究这些miRNA在T细胞活化和分化过程中的生物学功能有助于我们更好的调控适应性免疫应答。本课题用Exiqon miRNA基因表达谱芯片检测了用CD3抗体和CD28抗体双信号活化Jurkat细胞前后差异表达的miRNA,发现了5种miRNA在Jurkat细胞活化后表达下调。实时定量检测验证了芯片的结果。由于在已有的报道中发现,miR-181家族的成员miR-181a与T细胞和B细胞的发育有着密切的关系,在体外实验中也已证实anti-CD3抗体诱导的T淋巴细胞的活化会使miR-181的表达受到抑制。所以我们选择了miR-181c作为后续实验的miRNA分子。为了进一步确实miR-181c和人CD4+ T淋巴细胞活化的关系,我们用磁珠分离出正常健康人血中的CD4+ T淋巴细胞,实时定量证实在活化的人CD4+ T淋巴细胞中miR-181c的表达也是下调的。这与Exiqon miRNA基因表达谱芯片及Jurkat细胞检测结果一致。通过在网站http://microrna.sanger.ac.uk/和http://www.targetscan.org/上预测,发现T细胞活化通路的相关分子IL-2,CD69,IL1A,MAP3K3,MAP3K10,NFAT5的3’ UTR有miR-181c的结合位点,可能为miR-181c作用的靶分子。其中IL-2的3’ UTR有2个miR-181c的结合位点,而且在预测的靶分子的评分中排第二位,说明IL-2可能是miR-181c发挥作用的一个靶点。双荧光素酶报告系统检测的结果证实,miR-181c和IL-2的3’ UTR有较明显的结合。后续实验中,我们主要验证miR-181c和IL-2的关系。我们在体外用Jurkat细胞模拟人CD4+ T淋巴细胞。将miR-181c转染到Jurkat细胞内,用anti-CD3抗体和anti-CD28抗体双信号刺激活化Jurkat细胞,流式细胞术检测T细胞活化的表面标志分子CD25、CD69和CD154,发现和对照组相比,miR-181c可以部分抑制Jurkat细胞的活化。将miR-181c和IL-2 3’ UTR及其各种突变体共转染HEK293细胞,结果显示miR-181c对含有IL2 3’ UTR全长的荧光素酶报告基因的表达有比较明显的抑制作用,约为50%,只含有结合位点1的荧光素酶报告基因的表达被抑制了约20%,而只含有结合位点2的荧光素酶报告基因的表达被抑制了约2/3,说明结合位点2的抑制作用更强。为了进一步阐明IL-2是miR-181c的直接靶分子,还是miR-181c通过影响T淋巴细胞的活化间接影响了IL-2的表达水平,我们构建了IL-2的真核表达载体。pcDNA3-IL2CDS-UTR包含IL-2的编码区和3’ UTR,而pcDNA3-IL2CDS只包含IL-2的编码区。将这两种IL-2真核表达载体转染HEK293细胞,使得原本不表达IL-2的HEK293细胞上清高表达IL-2。同时转染合成的miR-181c分子和这两种IL-2真核表达载体到HEK293细胞中,ELISA检测HEK293细胞上清中的IL-2,转染miR-181c和pcDNA3-IL2CDS-UTR组的IL-2水平明显下调,而同时转染miR-181c和pcDNA3-IL2CDS组的IL-2水平没有显著变化,说明miR-181c可以通过结合IL-2 3’ UTR抑制IL-2蛋白水平的表达。说明IL-2是miR-181c的直接靶分子。将合成的miR-181c分子转染到人CD4+ T淋巴细胞内,用双信号刺激活化人CD4+ T淋巴细胞,流式细胞术检测T细胞活化的表面标志分子CD25、CD69和CD154,同样发现miR-181c可以部分抑制人CD4+ T淋巴细胞的活化。转染miR-181c的人CD4+ T淋巴细胞被活化24h后,和对照相比,上清中IL-2的水平明显下降,说明miR-181c可以抑制人CD4+ T淋巴细胞合成分泌IL-2。但是实时定量检测表明和未活化相比,活化组IL-2 mRNA水平显著上调,而转染miR-181c并没有下调IL-2 mRNA水平。在Jurkat细胞中观察到了同样的现象。说明由于miR-181c与IL-2 3’ UTR相互结合是不完全互补的,所以miR-181c只是抑制了IL-2 mRNA的翻译,而不是降解IL-2 mRNA。这与以往的文献报道相符合。分别给人CD4+ T淋巴细胞转染miR-181c和scramble miRNA(negative control, N.C.),用双信号刺激活化72h,用MTT检测其细胞增殖。转染miR-181c组活化72 h后,miR-181c抑制了人CD4+ T淋巴细胞的增殖,这可能与转染miR-181c后IL2的表达水平下调有关。以上实验结果表明:miR-181c与人CD4+ T淋巴细胞的活化有着密切的关系,它可以抑制人CD4+ T淋巴细胞的活化,同时也抑制人CD4+ T淋巴细胞表达IL-2分子。异位表达IL-2分子的实验证实可以直接下调IL-2分子的表达,IL-2分子是miR-181c的直接靶分子。miR-181c可以通过抑制IL-2的表达来抑制人CD4+ T淋巴细胞的活化。miR-181c是否还通过信号转导通路上的其他分子抑制T细胞的活化还有待于进一步的研究。更多还原

【Abstract】 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.更多还原