microRNA-212/132簇靶向调控MRGPRX2参与肥大细胞活化及过敏性哮喘发病的机制研究

Mastocytes have primarily been regarded as the crucial effector cells which cause harmful allergic reactions in allergic asthma, so the inhibition of mastocytes degranulation can effectively relieve asthma symptoms. The novel G protein-coupled receptor X2-MRGPRX2 can trigger mast cells degranulation and high level expression of MRGPRX2 was found in the lung tissue from asthmatic patients compared to non asthmatic. In our previous studies, it has been found that the MRGPRX2 knockout mice exerted a weaker inflammatory response to ovalbumin, while, the expression of miR-212/132(belong to the same gene cluster) which targeted MRGPRX2 reduced remarkably in the allergic asthma mouse model. It indicated that the downregulation of miR212/132 cluster may lead to the upregulation of MRGPRX2, and further propagate mast cells activation and degranulation, be involved in pathogenesis of allergic asthma. In this study, we will apply ELISA, qPCR, Western blot, reporter gene analysis, gene knock-out and knock-in, and so on, to explore the effects and mechanism of miR-212/132 in regulating MRGPRX2 expression on mastocytes activation, inflammatory cytokines expression and inflammation-related pathways, in clinical samples, in vitro and in vivo. Therefore, our research will provide an experimental and theoretical basis for the regulation of mastocytes activation by MRGPRX2 in the treatment of allergic asthma and potential clinical targeted therapy for allergic asthma.

肥大细胞是过敏性哮喘发生中关键的效应细胞，抑制肥大细胞脱颗粒能够有效降低炎症反应进而缓解哮喘症状。新型G蛋白耦联受体MRGPRX2，在哮喘患者肺组织中高表达，激动后可引起肥大细胞脱颗粒。我们前期研究发现，MRGPRX2基因敲除小鼠对卵清蛋白诱发的气道炎症明显减弱；而在过敏性哮喘小鼠模型中，靶向该基因的miR-212/132（两者同属一个基因簇）表达均显著降低，提示miR-212/132簇下调导致MRGPRX2表达上调，促进肥大细胞活化及脱颗粒，从而参与过敏性哮喘发病。本研究拟应用ELISA、qPCR、蛋白质印迹、报告基因、基因敲除与敲入等技术在临床、细胞和动物模型水平分析miR-212/132调控MRGPRX2表达在肥大细胞活化、炎性细胞因子表达及炎症相关信号通路中的作用机制，为通过MRGPRX2抑制肥大细胞活化治疗过敏性哮喘提供实验及理论依据，为寻找过敏性哮喘潜在治疗靶点提供新的认识。

microRNAs (miRNAs)作为转录后的主要调控因子，广泛参与人体生理活动的调节，并越来越多地用于疾病的诊断和治疗。miR-212/132已被证实参与多种慢性炎症疾病。肥大细胞(MCs)在过敏性疾病中起关键作用。本研究通过对临床样本的分析发现，哮喘患者血液中miR-212/132的含量明显下降，且与哮喘的潜在生物标志物MrgX2密切相关。. 已知C48/80可增加MC Ca2+内流并通过MrgX2激活MCs。体外研究中，脂质体转染miR-212/132，将miR212/132转移到LAD2细胞中48小时，然后用C48/80刺激。由于脱颗粒是活化MCs的标志，我们检测了β-己糖苷酶、组胺和炎症因子(如TNF-α、mapp -2和CCL-2)在LAD2细胞中的释放。结果显示，miR212/132剂量依赖性地降低LAD2中的Ca2+水平，显著降低了LAD2细胞的脱颗粒，以及TNF-α、MIP-2和CCL-2的水平。表明miR212/132可能通过MrgX2抑制c48 /80诱导的MC激活。表明miR212/132抑制MrgX2，发挥抗过敏的作用。. 在哮喘小鼠体内，发现转染miRNA后，miR212/132显著降低了OVA刺激的哮喘小鼠的肺组织炎症，包括肺组织炎症浸润、粘液分泌、胶原含量和脱颗粒MC比值；还显著抑制了BALF和血清中的趋化因子和细胞因子水平。通过RNAseq筛选得到二磷酸腺苷核糖化因子鸟苷酸激酶1(Adenosine diphosphate ribosylation factor guanylate kinase 1,ASAP1)，ASAP1是顶端连接复合体（apical junction complex ,AJC）的关键蛋白。用PT-PCR的方法证实，ASAP1在体外C48/80诱导的MCs激活和体内OVA诱导的肺部炎症中发挥着关键作用。miR212/132转染MCs48 h后，MrgX2表达显著升高。. 本研究证实，miR-212/132通过ASAP1抑制C48/80诱导的MCs激活和OVA诱导的肺部炎症，MrgX2参与了这一过程。为过敏性哮喘及其治疗提供了新的视角。.