miR-93多靶调控免疫功能的作用机制及其防治排斥反应的效果研究

Currently, great attention has been drawn on the regulatory networks of miRNAs in immune responses. However, there is still seldom relevant report concerning the involvement of miRNA in transplant rejection, whether miRNAs could serve as a therapeutic target to effectively treat rejection. Previously, we performed a miRNA microarray analysis among dendritic cells under varying maturity stages and found a promising miR-93, whose overexpression significantly decreased the production of cytokines in macrophages challeged by VSV virus. Furthermore, we demonstrated that miR-93 could markedly inhibit IL-2 signaling pathway by targeting on JAK1 and RASD1. As JAK-STAT pathway plays a critical role in the signal transduction of IL-2, in this study, we aim to further investigate the potential role of miR-93 in the prevention of transplant rejection, and the underlying mechanism involved in the rejection immunological responses. We plan to investigate the in vivo anti-rejection effect of miR-93LNA in murine cardiac transplant models, and how in vivo administered cholesterol-conjugated miR-93 affects the graft survival and rejection in rat liver transplantation. Next, we would construct miR-93 sponge mice model to examine how the miRNAs targeting JAK1 affect the immune responses and explore the possibility of miR-93 as an anti-rejection therapeutic target. Through this study, we would possibly clarify the mechanism by which miR-93 regulates immune responses and provide experimental data for the exploration of miRNA as feasible treatment of rejection.

miRNA对免疫细胞的调控是免疫学研究的热点。但有关miRNA是否参与移植排斥、体内作用效果如何、是否可作为分子靶点有效治疗排斥反应仍有待探究。我们通过对DC活化前后miRNA测序，发现miR-93可抑制DC产生TNF，并发现其可作用于多个重要分子的基因靶点如JAK1及Tnfaip1等；考虑到JAK-STAT在IL-2等CK信号传递中的重要性及miR-93调控多个重要靶基因，本申请拟研究miR-93抗排斥反应的效果，探讨miR-93多靶基因调控免疫功能的作用机制。构建慢病毒及miR-93sponge小鼠，研究过表达及低表达miR-93调控JAK1等多个靶基因后对DC、T细胞的调控作用和功能改变；采用心脏移植模型，研究过表达及低表达miR-93机体对同种抗原反应性及对排斥反应的影响；探讨miRNA作为排斥反应治疗的可行性，为认识miRNA调控免疫反应机制及发现治疗排斥反应的miRNA提供数据.

miRNA对免疫细胞的调控是免疫学研究的热点。但有关miRNA是否参与移植排斥、体内作用效果如何、是否可作为分子靶点有效治疗排斥反应仍有待探究。我们对DC细胞活化前后进行miRNA芯片测序分析，筛选出DC活化后表达降低最为显著miR-93等。通过双荧光报告系统，提示miR-93可作用于JAK1蛋白等靶点，能显著抑制IL-2等细胞因子的信号传导，具有明显的免疫调节及免疫抑制作用；进一步研究miR-93对排斥反应防治作用的效果，并探讨miR-93调控免疫功能的作用机制。结果发现：1）转染miR-93组DC表达的CD40、CD80、CD86和Ia明显低于对照组，其分泌TNF-α、IL-6、IL-12、IFN-β的能力下降。2）在基因水平上干扰JAK1的表达能够下调DC细胞中TNF-α、IL-6 mRNA的表达，进一步验证JAK1是miR-93的作用靶点；3）miR-93能够抑制DC细胞介导的抗原特异性T细胞增殖，降低Th1型细胞因子的分泌；4）采用同种小鼠心脏移植、大鼠肝脏移植模型发现体内高表达miR-93可以抑制移植排斥，延长移植物存活期。鉴于miR-93靶向JAK1产生的生物学效应可能与机体抗病毒免疫相关，我们进一步研究了miR-93在机体感染流感病毒（IAV）后的作用和机制。IAV感染后肺泡上皮细胞(AT2)表达miR-93通过抑制JAK/STAT信号途径促进IAV病毒表达的复制；体内给予antagomiR-93可显著抑制IAV的感染，显著降低IVA感染小鼠的死亡率。我们的研究结果不仅揭示了miR-93在基因表达调控水平控制DC与Th细胞为基础的免疫识别与排斥反应产生的机制，也揭示了该miRNA在机体遭受外界病毒感染时如何发挥调控作用的分子机制。