miR-155/ STAT3调控Th17细胞参与急性GVHD的机制研究

Acute graft-versus-host disease(aGVHD) is the most serious complication of hematopoietic stem cell transplant, the mechanism is complex. Research declared that Th17 cell aggravated aGVHD, our previous study showed that miR-155 expression is up-regulated in the serum from patients with aGVHD. Serum IL-17 level and the proportions of Th17 cell are positively correlated with miR-155 level in aGVHD patients. New research showed that STAT3 signaling and resultant Th17 tissue accumulation is closely associated with acute GVHD. STAT3 ChIP-qPCR showed that enrichment of STAT3 around the promoter region of miR-155. We speculated Th17 cell may participate in aGVHD pathophysiology by miR-155/STAT3 signal. In this project, we analyze miR-155、STAT3 among Th17 cell from more allogeneic HCT recipients. In vitro experiments, we analyze the expression profile of miR-155 among T cell, and further a functional link between STAT3 and miR-155 in the regulation of Th17 differentiation. We use genetic defect miR155 mouse model 、CD4-STAT3 KO mouse model and haploid transplantation mouse model as the experimental platform, explore the molecular mechanism of STAT3 regulating miR155 expression in Th17 cells in aGVHD. This study will help understand the role of miR155/STAT3 regulation in Th17 cells in aGVHD situation, in order to elucidate the pathogenesis of aGVHD, provide theoretical basis for clinical treatment of aGVHD.

急性移植物抗宿主病（aGVHD）是异基因造血干细胞移植最致命的并发症，病因复杂。我们的研究发现，microRNA155（miR155）在aGVHD发生时显著升高，与Th17数量及血浆IL-17a表达呈正相关。感染编码信号转导和转录激活因子3（STAT3）在miR155基因启动子部位异常丰富，与aGVHD靶器官中Th17聚集密切相关。由此推测：miR155/STAT3途径可通过调控Th17参与aGVHD。本项目拟①扩大aGVHD临床数据明确miR155/STAT3与Th17的相互关系；②体外实验探讨miR155对Th17的调控是否依赖STAT3途径；③以miR155基因缺陷和CD4-STAT3 KO鼠的aGVHD模型，体内验证miR155/STAT3对aGVHD的作用机制。本研究旨在明确miR155/STAT3调控的Th17在aGVHD中的作用，以阐明疾病的发病机理，为靶向治疗提供理论依据。

课题旨在探讨异基因移植后急性移植物抗宿主病（aGVHD）的致病机制，重点是相关miRNA、细胞因子及免疫调节细胞对aGVHD的诊断及预后机制。主要的成果包括，（1）扩大临床研究的样本量，分析异基因移植后患者体内miR-155-5p等多种移植免疫相关的miRNA的表达，从基因标志物层面探讨miRNA与aGVHD、Th17细胞、细胞因子的相关性，结果显示miR-155-5p，miR-181a-3p在GVHD患者的血浆中高表达，与GVHD严重程度呈正相关。IFN-γ、IL-17a、IL-9与miR-155-5p的表达呈正相关，IL-4与miR-155-5p呈负相关，IL-2、IL-22、IL-17a与miR-181a的表达呈正相关，IL-13与miR-181a呈负相关，提示不同miRNA所影响的细胞因子不尽相同。（2）从细胞标志物层面，分析细胞亚群Th17、Tc17在移植物抗宿主病中的变化，发现Th17、Tc17在aGVHD中显著增加，Th17在肝脏aGVHD中发挥主导作用，miR-155-5P的表达与Th17呈正相关，miR-181a-3P的表达与Th17、Tc17呈正相关。（3）从蛋白标志物层面，分析多种细胞因子在急性移植物抗宿主病中的表达，新发现Flt-3L、LAG-3两种因子在aGVHD中显著升高，有较好的敏感性和特异性。建立一个多因子(IL-7、IL-17a、IP-10、Flt-3L、LAG-3)诊断Panel，结果显示panel具有更高的特异性和敏感性，优于单个因子，可提高预测和监控aGVHD的准确性。（4）探讨了miR-155-5p是否通过调控STAT途径来影响Th17的分化。靶基因预测未发现miR-155与STAT3直接关联，但能靶向到与STAT1相关的PIAS1。文献报道STAT1与STAT3可共同调节Th17分化，故我们进行了双荧光素酶验证试验，结果显示在293T细胞中，miR-155-5P mimics对于PIAS13′UTR-WT、PIAS13′UTR-MU1、PIAS13′UTR-MU2均无显著作用。miR-155-5P与STAT3、STAT1被证实无相关性，这一阴性结果提示我们可以另辟思路。本研究从基因水平、细胞水平、蛋白水平三个层面系统、立体的探讨了aGVHD致病过程中的机制，发现并验证高效、敏感的生物学标记物，为疾病的精准治疗提供有力保障。