Graves病MicroRNAs/mRNAs调控网络及其分子机制研究

Graves' disease (GD), an autoimmune disease, involves multiple factors in its pathogenesis under the genetic background. It was recognized that Th1/Th2 imbalance might contribute to its etiology several decades ago. In recent years, the discovery and establishment of several new T lymphocytes have brought a new era for investigation of the mechanism of GD. Our and others' researches have found that there are abnormal expressions of Treg, Th17, Tfh cells and their related factors Foxp, IL-17, IL-23, CXCR5, CD57 and so on. However, analysis of only a few cells or factors cannot fully explain this complex immune disorder. The gene chip can detect hundreds or thousands of gene expressions (mRNAs) simultaneously and help reveal the interactions between various cells and factors. MicroRNAs (miRNAs), a class of endogenous non-coding RNA, inhibits target gene expression. Abnormality of a specific miRNAs profile has been found in a variety of diseases including cancers and autoimmune diseases. This abnormally expressed miRNAs participate in the disease development through regulating the expression of key signaling molecules and regulating proliferation, differentiation and function of related cells. This study, based on observation of the spectrum of variable cytokine expression (mRNAs) and the spectrum of miRNAs expression in the diseased tissue of GD provided by gene chip technology aims to analyze the mutual regulation between miRNAs and target gene so as to further clarify the molecular and cellular mechanisms underlying the development and progression of GD. We believe that our investigation will also lay a foundation for establishment of new biological therapeutic approaches.

Graves病（GD）是多种因素参与的自身免疫性疾病，数十年前发现GD存在Th1/Th2平衡失调。近年来新型免疫细胞的发现开辟了GD研究的新纪元。我们及他人的研究发现GD存在Treg、Th17和Tfh细胞及相关因子Foxp3、IL-17、IL-23、CXCR5等的异常。但仅几个细胞或因子难以解释这种复杂疾病的免疫网络紊乱。基因芯片可同时检测成百上千个基因的表达（mRNA），且有助于分析各种细胞及因子的相互作用。MicroRNA（miRNA）是一种内源性表达的非编码小RNA, 对靶基因的表达起抑制作用。已证实肿瘤、免疫性疾病等存在miRNAs的表达异常。miRNAs通过调节靶信号分子的表达，调节细胞的增殖分化与功能，从而参与疾病的发生发展。本课题拟通过基因芯片技术观察GD病变组织的细胞因子表达谱（mRNA）、miRNA表达谱，以阐明GD的发生机理，为开辟生物治疗的新途径奠定基础。

本课题采用miRNAs芯片、mRNAs芯片技术，检测GD患者甲状腺组织差异miRNAs及mRNAs表达谱，并通过实时定量PCR(quantitative Real-time PCR, qRT-PCR)技术对部分芯片结果进行验证。Western Blot(WB)、体外细胞抑制实验及双荧光素酶报告基因检测对候选致病基因miR-660及其靶基因SDC1在GD中的表达及功能进行进一步分析，探讨miRNAs及其靶基因在GD发生、发展中的作用。. 实验证实：与正常对照相比，GD患者甲状腺组织差异表达miRNAs共23个，其中上调表达5个，下调表达18个（表达倍数比≥1.3且P<0.05）；差异表达mRNAs共1251个，其中上调表达805个，下调表达446个（表达倍数比≥2.0且P<0.001），且上调表达mRNAs多与免疫应答相关通路密切有关，如刺激物应答、防御应答、损伤及应激应答、白细胞和淋巴细胞活化等。（2）整合数据分析显示，miR-22及其靶基因外胚层发育不良蛋白（EDA）、甘氨酸脒基转移酶(GATM)、髓系/淋巴或混合谱系白血病易位蛋白4(MLLT4)，miR-183及其靶基因激酶锚定蛋白12(AKAP12)、多类型锌指蛋白12(ZFPM2)，miR-101及其靶基因前B细胞白血病同源盒3(PBX3)，miR-197及其靶基因软骨中层蛋白焦磷酸水解酶(CILP)，miR-660及其靶基因多配体蛋白聚糖1(SDC1)，可能是与GD发病有关的候选致病基因。（3）qRT-PCR及WB结果显示，SDC1高表达于GD患者甲状腺组织；miR-660以非专一、直接结合方式，轻度抑制SDC1表达，抑制率为11%。. 因此，异常表达的miRNAs、mRNAs参与了GD的发病。GD患者甲状腺组织差异表达miRNAs/mRNAs谱的建立，对于我们全面理解GD发病机理提供了数量庞大的理论依据。SDC1在GD患者甲状腺组织的高表达，提示其可能通过促炎效应参与了GD的发病，而miR-660作为调节SDC1表达基因组成员中的一种亦参与了GD的发生。