Serum Exosome MicroRNA in Systemic Sclerosis

摘要 系统性硬化症（SSc）是一种原因不明的全身性自身免疫性疾病，以进行性纤维化为特征 影响皮肤和内脏器官，导致严重残疾、多器官衰竭和高死亡率。肺 参与是SSc功能障碍和高死亡率的主要原因。SSc临床的主要未满足需求 治疗方法是缺乏经过充分验证的生物标志物来早期诊断和准确评估SSc 参与的严重程度和范围，或区分原发性雷诺现象患者与 雷诺现象有演变为SSc的风险。我们最近获得了强有力的初步结果表明， 从SSc患者血清中分离的外来体的microRNA（miRNA）分析可以提供独特且敏感的方法。 反映SSc程度和严重程度的生物标志物。基于这些结果，我们提出以下假设： 从SSc患者血清中分离的外来体的miRNA谱的无偏分析将允许以较高的分离效率分离外来体。 SSc的各种相关临床子集的准确性和敏感性程度，将显示与 SSc严重程度和肺纤维化累及范围，并将区分原发性雷诺综合征患者 来自雷诺现象患者的现象，这些患者携带血清抗核抗体（ANA）， 发展成SSc的风险很高。进一步的假设是：“在SSc中鉴定的差异表达的miRNA 血清外泌体在SSc组织纤维化的发病机制中起关键作用， 正常成纤维细胞或抑制SSc成纤维细胞的促纤维化表型。为了验证这些假设，我们将继续 以下具体目标： 具体目的1：使用下一代测序（NGS）技术鉴定以下基因的miRNA谱差异： A：患有近期发作的弥漫性SSc的患者相对于正常人 个体; B：Ssc相关肺纤维化患者与无肺受累的弥漫性SSc患者; C：原发性雷诺现象患者与ANA阳性雷诺现象患者。 具体目标2：检测是否最差异表达（上调或下调）的miRNA 目的1中鉴定的化合物对正常真皮和肺成纤维细胞发挥促纤维化作用或抑制纤维化 表型或SSc真皮和肺成纤维细胞。 本文提出的血清外泌体的无偏NGS miRNA分析是全新的，尚未应用于SSc 以前。这里鉴定的血清外泌体中存在的miRNA的差异可能会导致未来的发展， 非侵入性SSc生物标志物，将导致SSc患者护理标准的“转型变化”， 雷诺现象患者发展为SSc的高风险。我们进一步期望， 用差异表达最多的血清外泌体miRNAs进行的研究将鉴定新的分子机制， 参与SSc组织纤维化的途径。

ABSTRACT Systemic Sclerosis (SSc) is a systemic autoimmune disease of unknown origin characterized by progressive fibrosis affecting skin and internal organs leading to severe disability, multiple organ failure, and a high mortality rate. Pulmonary involvement is the leading cause of functional disability and high mortality in SSc. A major unmet need for SSc clinical management is the absence of well validated biomarkers that allow early diagnosis and accurate assessment of SSc severity and extent of involvement, or that differentiate patients with Primary Raynaud Phenomenon from patients with Raynaud Phenomenon at risk of evolving into SSc. We recently obtained strong Preliminary Results indicating that microRNA (miRNA) analysis of exosomes isolated from the serum of SSc patients may provide unique and sensitive biomarkers that reflect the extent and severity of SSc. Based on these results we propose the following hypothesis: “The unbiased analysis of the miRNA profile of exosomes isolated from SSc patient serum will allow to separate with a high degree of accuracy and sensitivity various relevant clinical subsets of SSc, will display a significant correlation with SSc severity and extent of pulmonary fibrotic involvement, and will distinguish patients with Primary Raynaud Phenomenon from patients with Raynaud Phenomenon harboring serum antinuclear antibodies (ANA) who are at high risk of evolving into SSc. A further hypothesis posits that: “The differentially expressed miRNA identified in SSc serum exosomes play a crucial role in the pathogenesis of SSc tissue fibrosis inducing profibrotic molecular events on normal fibroblasts or inhibiting the profibrotic phenotype of SSc fibroblasts.” To test these hypotheses we will pursue the following Specific Aims: SPECIFIC AIM 1: To identify employing Next Generation Sequencing (NGS) differences in the miRNA profile of exosomes isolated from the serum of the following individuals: A: Patients with recent onset diffuse SSc vs. normal individuals; B: Patients with SSc-associated pulmonary fibrosis vs. diffuse SSc patients without lung involvement; and C: Patients with Primary Raynaud Phenomenon vs. patients with ANA-positive Raynaud Phenomenon. SPECIFIC AIM 2: To examine whether the most differentially expressed (upregulated or downregulated) miRNA identified in Aim 1 exert either profibrotic effects on normal dermal and lung fibroblasts or inhibit the fibrotic phenotype or SSc dermal and lung fibroblasts in vitro. The unbiased NGS miRNA analysis of serum exosomes proposed here is entirely novel and has not been applied to SSc previously. The differences in miRNAs present in serum exosomes identified here may lead to the future development of non-invasive SSc biomarkers that will result in a “transformational change” in the standard of care for SSc patients and for patients with Raynaud Phenomenon at high risk of evolving into SSc. We further expect that the mechanistic studies performed with the most differentially expressed serum exosome miRNAs will identify novel molecular mechanisms and pathways involved in SSc tissue fibrosis.