CFlm25 mediated alternative polyadenylation regulates fibrosis in systemic sclerosis

系统性硬化症(SSC)是一种多系统的纤维性疾病，影响皮肤和各种内部 器官。肌成纤维细胞持续激活及其相关的过量细胞外基质蛋白(ECM) 口供是这种疾病的特征。解释这种过度纤维化反应的机制 仍然难以捉摸。尽管它的死亡率和发病率很高，但目前还没有FDA批准的治疗纤维化的药物 硬膜外血肿并发症。我们最近的工作表明，一种称为替代的RNA处理机制 多聚腺苷酸化(APA)是通过3‘UTRTail上调真皮纤维化中促纤维化基因表达的关键 缩短关键文字记录。最近的证据表明，APA的一个关键调节因子是切割因子Im 25(CFIM25)。CFIm25缺失导致3‘非编码区缩短。截断的3‘非编码区尾巴通常会缺少 MicroRNA结合部位，逃避microRNA介导的基因抑制。我们的初步数据显示 CFlm25在SSC患者纤维化皮肤和小鼠皮肤纤维化模型中表达下调。击倒 正常皮肤成纤维细胞中的CFIm25足以促进关键促纤维化基因的3‘非编码区缩短。 此外，中心性纤维化细胞因子转化生长因子β通过上调miR-203抑制CFIM25的表达。 总体而言，我们的数据揭示了一种新的机制，转化生长因子β介导的cfim25下调导致3‘ UTR缩短和促纤维化因子和ECM的过度产生，参与了发病机制 SSC.基于这一初步数据，本项目的主要假设是转化生长因子β和miR-203 成纤维细胞中CFIM25表达下调，通过上调促纤维化基因导致真皮纤维化 细胞外基质通过3‘非编码区缩短。这一假设将在以下具体目标中得到检验： 目的I：明确CFIm25缺失在小鼠皮肤纤维化模型中对成纤维细胞的特异性贡献。 这一目标将阐明CFIm25缺失对关键的纤维化途径的下游影响。目标二： 确定CFIm25下调的机制并评估其治疗潜力 真皮纤维化的靶点。转化生长因子β介导的miR-203下调及其机制 我们将阐明miR-203对CFIm25的抑制作用。这一目标将描述导致以下情况的上游事件 CFIm25耗尽，并将确定潜在的治疗靶点。目的III：在SSC人类中鉴定CFIm25 皮肤/成纤维细胞，并鉴定APA失控的纤维化基因。系列真皮成纤维细胞与皮肤 来自早期SSc患者和匹配的对照组的样本将使用一种新的RNA测序进行检查 技术这一目标将首次为CFIm25介导的APA提供一个公正的、纵向的观点 纤维性疾病的侧写。 这项拟议的研究首次将CFIM25介导的3‘非编码区缩短与真皮纤维化联系起来。这 可以导致发现一种关键的机制，该机制可以放大SSC的纤维化反应，并最终 确定一种全新的治疗这种潜在破坏性疾病的目标。

Systemic sclerosis (SSc) is a multi-system, fibrotic disease that affects the skin and a variety of internal organs. Persistent myofibroblast activation and associated excessive extracellular matrix protein (ECMs) deposition are hallmarks of the disease. The mechanisms that account for this excessive fibrotic response remain elusive. Despite its high mortality and morbidity, there are no FDA approved medications for fibrotic complications of SSc. Our recent work indicates that a RNA-processing mechanism known as alternative polyadenylation (APA) is critical for the upregulation of profibrotic genes in dermal fibrosis through 3’ UTR tail shortening of key transcripts. Recent evidence demonstrated that a key regulator of APA is Cleavage factor Im 25 (CFIm25). The CFIm25 deletion leads to 3’ UTR shortening. A truncated 3’ UTR tail will often lack microRNA binding sites and evades the microRNA-mediated gene repression. Our preliminary data indicate CFlm25 is downregulated in fibrotic skin of SSc patients and in murine dermal fibrosis models. Knockdown of CFIm25 in normal skin fibroblasts is sufficient to promote the 3’ UTR shortening of key profibrotic genes. Moreover, the central fibrotic cytokine TGFβ suppresses CFIm25 expression through miR-203 upregulation. Overall, our data uncovered a novel mechanism by which TGFβ mediated CFIm25 downregulation leads to 3’ UTR shortening and the over-production of profibrotic factors and ECMs, and contributes to the pathogenesis of SSc. Based on this preliminary data, the main hypothesis of this project is that TGFβ and miR-203 downregulate CFIm25 in fibroblasts, resulting in dermal fibrosis by upregulation of profibrotic genes and ECMs through 3’ UTR shortening. This hypothesis will be examined in the following specific aims: Aim I: Define the fibroblast specific contribution of CFIm25 depletion in dermal fibrosis murine models. This aim will elucidate the downstream effects of CFIm25 depletion on key fibrotic pathways. Aim II: Determine the mechanisms for CFIm25 downregulation and assess their potential as therapeutic targets in dermal fibrosis. The mechanisms for TGFβ mediated miR-203 downregulation and subsequent CFIm25 repression by miR-203 will be elucidated. This aim will characterize the upstream events leading to CFIm25 depletion and will identify potential therapeutic targets. Aim III: Characterize CFIm25 in SSc human skin/fibroblasts and identify fibrotic genes dysregulated by APA. Serial dermal fibroblasts and skin samples from patients with early SSc and matched controls will be examined using a novel RNA sequencing technology. This aim will provide for the first time an unbiased, longitudinal view of CFIm25 mediated APA profile in a fibrotic disease. The proposed research links for the first time the CFIm25 mediated 3’ UTR shortening to dermal fibrosis. This can lead to discovering a key mechanism that amplifies the fibrotic response in SSc and ultimately to identifying an entirely novel target for treatment of persons with this potentially devastating disease.