从 LncRNA AK005410 通过 miR-145a-5p/RUNX1 调控Treg探讨葡萄膜炎的发病机制与治疗

Uveitis is an autoimmune eye disease of high probability of causing blind, which is mediated with Treg/Teff imbalance. Increasing the number of Treg or enhancing its function is the key to successful treatment. Previous studies indicate that LncRNAs could act as sponge to compete for miRNAs which are supposed to binding to mRNA targets, thus regulate protein-coding and participate in the progress of diseases. We have identified differential expressed LncRNA (AK005410 ↑) and miRNA (miR-145a-5p ↓)between Experimental Autoimmune Uveitis and normal control via microassays and comprehensive analysis, moreover, LncRNA AK005410 has 3 binding sites and could interact with miR-145a-5p . After searching for predicted miR-145a-5p targets in several online databases, and we identified RUNX1 which was critically involved in biological process of T cell differentiation , was the target of miR-145a-5p. Here, we aim at investigate AK005410/miR-145a-5p/RUNX1 in the pathogenesis of uveitis via regulating Treg cells, and provide scientific evidence for diagnosis and therapy.

葡萄膜炎是Treg/Teff失衡介导的自身免疫性、高致盲眼病。提高Treg数量或增强其功能是治疗关键。既往研究显示，LncRNAs可作为海绵吸附体竞争性结合miRNAs，调控其靶基因mRNA编码蛋白能力，参与疾病发生发展。我们前期通过基因芯片筛选出自身免疫性葡萄膜炎（EAU）小鼠和正常小鼠差异表达的LncRNAs和miRNAs，进行生物信息分析及功能鉴定，确定人鼠同源目标LncRNA AK005410（↑）,其序列中与候选miRNA miR-145a-5p（↓）有3个结合位点且能竞争性结合；且miR-145a-5p的靶基因RUNX1,具有调控T细胞分化的功能。本课题拟通过基因敲除、慢病毒转染、siRNA沉默等技术，探讨LncRNA AK005410/miR-145a-5p/RUNX1通过调节Treg分化、功能和稳定性参与葡萄膜炎发生发展机制，并进行临床验证，为葡萄膜炎的诊疗提供科学依据。

项目背景：. 葡萄膜炎是临床上多见、病因不明且易复发的的高致盲性眼病，Treg／Th17细胞失衡被认为是葡萄膜炎发病的核心环节，但引起失衡的原因仍不清楚。为进一步探讨细胞失衡的机制，从而寻找恢复细胞平衡的方法是治疗该病和改善预后的关键。ncRNA能通过调控下游靶基因mRNA的编码蛋白能力，参与多种自身免疫性疾病发生发展。然而在葡萄膜炎中ncRNA的表达以及作用机制尚不明确。..主要研究内容：.1）葡萄膜炎患者和小鼠中LncRNA AK005410、miR-145a-5p的表达情况。.2）通过靶基因预测明确LncRNA AK005410、miR-145a-5p与RUNX1相互作用关系。.3）体外实验证明miR-145a-5p对T细胞作用。.4）体内实验证明miR-145a-5p对T细胞和葡萄膜炎的作用。.5）通过葡萄膜炎患者临床样本，初步分析Th细胞亚群。..重要结果：.1）葡萄膜炎小鼠外周淋巴结细胞和视网膜细胞中LncRNA AK005410表达上调、miR-145a-5p下调；葡萄膜炎患者外周血中LncRNA AK005410表达上调、miR-145a-5p表达下降。.2）靶基因预测明确LncRNA AK005410、miR-145a-5p与RUNX1相互作用关系。.3）体外上调的miR-145a-5p引起Treg细胞数量增多、诱导率提高、功能增强；引起Th17数量降低；.4）体内实验证明miR-145a-5p上调的Treg对葡萄膜炎有缓解作用；.5）通过葡萄膜炎患者临床样本，初步分析Th细胞亚群；.6）通过动物和体外试验发现PDE4抑制剂、钙离子通道阻滞剂、载脂蛋白A1、阿奇霉素和特立氟胺通过不同下游通路，恢复Treg／Th17细胞平衡，缓解葡萄膜炎。..科学意义：. 深入探讨ncRNAs的作用扩大了对葡萄膜炎发病机制的认识，为进一步葡萄膜炎的治疗提供新的理论基础，同时上调miR-145a-5p对葡萄膜炎有缓解作用，对应用于临床葡萄膜炎患者有较好的前景。