MiR-338-3p在天疱疮发病中通过调节Th1/Th2细胞调控抗Dsg3抗体的作用及机制研究

Pemphigus vulgaris （PV） is a potentially fatal autoimmune disease caused by anti-Dsg3 antibody. But its upstream mechanism of the antibody production remains uncertain. It has been found that the onset of PV is associated with the abnormal expression of susceptibiligy genes and the dysfunction of Th1/Th2 cells in peripheral blood may be a pivotal factor for the production of anti-Dsg3 antibody, which suggests that the mechanisms of the balance maintenance of Th1/Th2 cells and the production regulation of anti-Dsg3 antibody need to be clarified urgently. Our previous study has found that the expression of miR-338-3p was abnormally elevated in PBMC of peripheral blood from patients with PV analyzed by microRNA microarray screening, which has been further proved by the preliminary small sample experiments, indicating that miR-338-3p might play an important role in the pathogenesis of PV. In this project, we intend to analyze the relationship between the expression of miR-338-3p and the disease condition firstly. Then the function and mechanism of miR-338-3p on the balance of Th1/Th2 cells and the production of anti-Dsg3 antibody will be explored by performing miR-338-3p overexpression experiments. Finally, miR-338-3p KO Dsg3-/- and Rag-2-/- mouse models will be used to verify the role and mechanism of miR-338-3p in vivo. The study findings will help to explain the pathogenesis of PV and provide a theoretical basis for future target gene therapies.

寻常型天疱疮（PV）是一种由抗Dsg3抗体诱发的有潜在致命危险的自身免疫病，但该抗体上游形成机制未明。现已发现PV发病与易感基因异常表达有关，且外周血Th1/Th2细胞功能紊乱可能是抗Dsg3抗体产生的重要因素，提示Th1/Th2细胞平衡维持及抗Dsg3抗体产生调节的机制急待明确。本课题前期经PV外周血单个核细胞microRNA芯片筛查发现miR-338-3p在PV中表达异常升高，并经小样本初步检测验证，提示miR-338-3p在PV发病机制中可能起重要作用。本项目拟先分析miR-338-3p表达与PV病情的相关性，再经miR-338-3p细胞转染实验探索其对Th1/Th2细胞功能平衡和抗Dsg3抗体产生的作用及机制，最后利用miR-338-3pKO Dsg3-/-小鼠及Rag-2-/-小鼠验证miR-338-3p在体的作用及机制，为进一步阐明PV发病机制和将来的基因靶向治疗提供理论基础。

寻常型天疱疮（PV）是一种由抗Dsg抗体诱发的有潜在致命危险的自身免疫病，外周血Th1/Th2细胞功能紊乱是抗Dsg抗体产生的重要因素，Th1/Th2细胞平衡维持及抗Dsg抗体产生调节的机制亟待明确。本课题前期研究发现miR-338-3p在PV中表达异常升高。本项目扩大样本量证实miR-338-3p在PV患者PBMCs和CD4+T细胞中表达均显著上升，其上升水平与疾病严重程度呈正相关，提示miR-338-3p可作为PV疾病诊断和病情严重程度评估的潜在有效标记物。体外过表达PBMCs的miR-338-3p可引起Th1细胞相关因子表达下降，Th2相关细胞因子表达升高；抑制PBMCs的miR-338-3p结果相反，这提示miR-338-3p可通过促进Th1/Th2细胞免疫平衡趋向Th1细胞，促进PV的发生发展。进一步利用CD4+T细胞探讨miR-338-3p调控Th/Treg细胞免疫平衡机制，过表达CD4+T细胞的miR-338-3p可引起RUNX1、FOXP3基因表达下降，而抑制miR-338-3p可导致RUNX1、FOXP3基因表达升高。RUNX1蛋白是FOXP3基因表达的重要转录因子，正向调控FOXP3的表达，而FOXP3是CD4+T细胞亚群Treg细胞的特异性转录因子，在Treg细胞介导的免疫抑制作用中发挥重要作用。TargetScan工具预测RUNX1蛋白是miR-338-3p的靶蛋白，荧光素酶报告基因实验证实miR-338-3p通过靶向抑制CD4+T细胞内RUNX1的表达以降低FOXP3的表达，从而使Treg细胞的免疫抑制功能受损，促进PV患者体内Th/Treg细胞免疫失衡和疾病发生发展。进一步研究PV患者Treg/Th2/Th7免疫失衡的分子机制，发现PV患者外周Th2/Treg细胞介导的免疫平衡倾向于Th2细胞，且Th17细胞异常活化。PV患者外周CD4+T细胞的PI3K/AKT/mTOR/P70S6K、ERK/MAPK信号通路表达增强，抑制mTOR信号通路的活性可逆转Th2/Treg细胞比例失衡，而抑制ERK/MAPK信号通路的活性可抑制Th17细胞的分化。上述研究较为深入和全面地探明miR-338-3p参与调节PV外周Th/Treg免疫失衡的机制，以及阐明信号通路异常在PV外周Th/Treg免疫失衡的调控作用，为将来PV靶向基因治疗提供理论依据。