Role of microRNA in Resveratrol-mediated suppression of mast cell-driven inflammation

摘要 过敏性疾病是美国第五大慢性病。引起过敏的一个主要因素 包括哮喘在内的炎症是由肥大细胞产生的前列腺素D2(PGD2)，这种细胞类型负责 免疫球蛋白介导的即刻超敏反应。因此，靶向于肥大细胞中的花生四烯酸途径 细胞抑制PGD2的生物合成是显著限制过敏性炎症的策略之一。最近，我们 发现白藜芦醇，一种天然的植物来源的多酚，选择性地抑制依赖IgE的PGD2 从人类皮肤肥大细胞产生。我们进一步表明，白藜芦醇抑制FcεRI诱导的表达 环氧合酶2(COX-2)是花生四烯酸途径中的一个关键酶，直接参与PGD2 生物合成。本研究的中心问题是：白藜芦醇如何抑制FcεRI诱导的COX-2表达 肥大细胞产生PGD2？有趣的是，miR-155被认为是COX的正向调节因子。 2在不同肿瘤、巨噬细胞、气道平滑肌中的表达，以及与过敏性哮喘严重程度的关系 老鼠。事实上，我们的miRNA阵列分析和qRT-PCR验证研究显示， FcεRI交联后人原代肥大细胞中MIR-155和COX-2的表达此外，我们发现， 白藜芦醇显着抑制FcεRI诱导的miR-155和COX-2的表达。vt.给出 这些数据表明，miR-155针对的是COX-2的抑制子。我们的 在计算机途径分析中已经发现了几种COX-2的负调控因子，如ATF3，SOCS-1，SHIP-1， 和PPARG，作为miR-155在人和小鼠肥大细胞中的潜在靶点。因此，我们假设 白藜芦醇通过调节肥大细胞miR-155的表达抑制变应性炎症 增加抑制物的诱导，从而减少COX-2的表达和PGD2的生物合成。 我们的研究将：(1)研究白藜芦醇对FcεRI诱导的小鼠脑内MIRNA表达的影响。 人和小鼠的原位成熟肥大细胞，(2)鉴定miR-155靶向的COX-2抑制物，以及 明确白藜芦醇抑制FcεRI诱导的COX-2表达和PGD2生物合成的机制 以及(3)研究饮食中白藜芦醇对气道重塑和高密度脂蛋白的影响。 过敏性哮喘肥大细胞依赖模型的反应性，并识别相关的miRNAs。要定义 MiR-155-5p在过敏性哮喘中的作用及白藜芦醇抑制疾病发展的作用 在我们的模型中还将使用miR-155-5p转基因(TG)和基因敲除(KO)小鼠。总体而言，这项研究将带来新的 MiR-155在白藜芦醇选择性抑制Fc、ε、RI诱导的COX-2和PGD2中的作用 在肥大细胞中产生，从而减轻过敏性炎症。

ABSTRACT Allergic disease is the fifth leading chronic disease in the United States. A major contributing factor to allergic inflammation including asthma is Prostaglandin D2 (PGD2) produced by mast cells, the cell type responsible for IgE-mediated immediate hypersensitivity reactions. Therefore, targeting the arachidonic acid pathway in mast cells to inhibit PGD2 biosynthesis is one strategy to significantly limit allergic inflammation. Recently, we discovered that Resveratrol, a natural plant-derived polyphenol, selectively inhibited IgE-dependent PGD2 production from human skin mast cells. We further showed that Resveratrol inhibited FcεRI-induced expression of cyclooxygenase 2 (COX-2), a key enzyme in the arachidonic acid pathway that is directly involved in PGD2 biosynthesis. The central question of this study is: how does Resveratrol inhibit FcεRI-induced COX-2 expression and PGD2 production in mast cells? Interestingly, miR-155 has been implicated as a positive regulator of COX- 2 expression in different cancers, macrophages, airway smooth muscle, and in the severity of allergic asthma in mice. Indeed, our miRNA array analysis and qRT-PCR validation studies revealed a positive correlation between miR-155 and COX-2 expression in human primary mast cells following FcεRI crosslinking. Moreover, we found that Resveratrol significantly inhibited FcεRI-induced expression of miR-155 expression as well as COX-2. Given that miRs negatively regulate target genes, these data suggest that miR-155 targets a repressor of COX-2. Our in silico pathway analysis has identified several negative regulators of COX-2 such as ATF3, SOCS-1, SHIP-1, and PPARG, as potential targets of miR-155 in human and murine mast cells. Thus, we hypothesize that Resveratrol inhibits allergic inflammation by regulating the expression of miR-155 in mast cells leading to increased induction of the repressors and consequently diminishing COX-2 expression and PGD2 biosynthesis. Our study will (1) Characterize the effect of Resveratrol on the FcεRI-induced miRNA expression profile in in situ-matured mast cells from human and mouse, (2) Identify the COX-2 repressor targeted by miR-155, and define the mechanism by which Resveratrol inhibits FcεRI-induced COX-2 expression and PGD2 biosynthesis in mast cells, and (3) Characterize the effects of dietary Resveratrol on airway remodeling and hyper- responsiveness in a mast cell dependent model of allergic asthma, and identify associated miRNAs. To define the role of miR-155-5p in allergic asthma and the efficacy of Resveratrol in inhibiting disease development, we will also use miR-155-5p transgenic (Tg) and knockout (KO) mice in our model. Overall, this study will shed new light on the role of miR-155 in the ability of Resveratrol to selectively inhibit FcεRI-induced COX-2 and PGD2 production in mast cells, thereby, attenuating allergic inflammation.