EZH2去甲基化正向调控JAM-A在显微镜下多血管炎中的分子机制

Pathogenesis of microscopic polyangiitis (MPA) has been a hot topic in the researches of autoimmune diseases. Our previous study found in vascular walls and immune cell surfaces of patients with MPA, expression of the junctional adhesion molecule A (JAM-A) was significantly increased, and this is largely regulated by Histone-lysineN-methyltransferase2 (EZH2). Combining international and domestic researches, we make a hypothesis: in MPA, EZH2 is up regulated in CD4+T cell, which depletes the activity of H3K27me3. This demethylation process promotes adhesion between vascular endothelial cells (VEC) and immune cells, and promotes the release of inflammatory factors and specific autoantibodies, resulting in the inflammation of vascular walls. This study intends to carry out: ①exploring the regulatory role of EZH2 on JAM-A mediated by demethylation ; ②exploring effects of EZH2 on adhesion, migration, proliferation and secretion of CD4+T cell and the underlying molecular mechanism; ③ exploring upstream molecular mechanism of demethylation in MPA through sequencing and bioinformatic analysis. This study is based on the clinical findings and we aim to study the regulation mechanism of MPA vascular inflammation in deep, to better understand the precise regulatory network of MPA vascular inflammation, and to provide a new perspective for MPA intervention.

显微镜下多血管炎(MPA)的发病机制一直是自身免疫疾病的研究热点，我们前期研究发现，MPA患者血管内壁及免疫细胞表面的连接粘附分子A(JAM-A)的表达与组蛋白赖氨酸N甲基转移酶(EZH2)介导的去甲基化作用密切相关。因此我们结合国内外研究假设：MPA中CD4+T细胞的EZH2上调，通过H3K27me3异构和失能削弱DNA的甲基化从而上调JAM-A，进一步诱导了免疫细胞与血管内皮细胞的粘附，促进自身抗体及炎症因子的产生和释放导致血管炎症。本研究拟开展：①明确在MPA中EZH2通过DNA去甲基化对JAM-A的功能调控；②探究EZH2去甲基化调控CD4+T细胞粘附、迁移、增殖、分泌的作用机制；③通过测序和生信分析探寻MPA中影响EZH2表达的上游分子机制。本研究立足临床深入研究MPA血管炎症的调控机制，以期为深入理解MPA血管炎症反应的精准调控网络提供新视角，为靶向干预MPA提供新依据和思路。

该课题按照原定计划，完成了人微血管内皮细胞系(HMEC)中过表达/抑制JAM-A对细胞增殖、迁移能力影响的细胞实验。主要研究内容为：①对稳定培养的人微血管内皮细胞进行病毒转染，并用嘌呤霉素进行细胞筛选，倒置显微镜下观察转染效率；②使用飞捷生物RNA fast200总RNA快速抽提试剂盒提取转染后的HMEC的RNA，通过定量PCR(Q-PCR)比较未转染细胞及转染后细胞JAM-A的表达情况；③流式细胞检测术检测转染后细胞的凋亡及周期；④ Transwell小室和划痕实验观察转染后的细胞迁徙、侵袭能力的变化。研究结果显示：JAM-A对HMEC的增殖具有正向调控的作用，而过表达或抑制JAM-A均可以增快HMEC的迁移，可能是AAV治疗的一个新的生物靶点。