The Altered DNA Methylome as a Determinant of Variable Disease Progression in IPF

描述(申请人提供)：特发性肺纤维化(IPF)是一种破坏性的肺部疾病，目前尚无有效的治疗方法。尽管中位存活率很低，但个别患者的预后是可变的和不可预测的，一些患者保持稳定，另一些患者迅速恶化。解释患者临床变异性的机制 病程尚不清楚。成纤维细胞是纤维化的主要效应细胞，其持续和旺盛的活性被认为有助于IPF的进行性。IPF成纤维细胞表现出即使在多次细胞传代后仍在培养中持续存在的基因表达变化，这表明表观遗传修饰可能是这些基因表达差异的原因之一。在特发性肺间质纤维化成纤维细胞中已发现部分基因的DNA甲基化改变，但对特发性肺间质纤维化的DNA甲基化异常及其对疾病进展的贡献的全球评估从未被评估过。本项目的目的是探讨IPF细胞全基因组DNA甲基化和转录改变如何影响IPF的临床变异性，并确定特定的抗纤维化和促纤维化介质前列腺素E2(PGE2)和转化生长因子-�1(TGF1)如何影响IPF中DNA甲基化模式的改变。中心假设是IPF患者的成纤维细胞在多种细胞外信号的反应下，DNA甲基组发生了明显的变化，这些不同的甲基化模式导致了成纤维细胞的异质性基因表达谱和IPF患者不同的临床进展速度。该项目有两个具体的目标：1)研究成纤维细胞中DNA甲基化的变化如何促进成纤维细胞基因表达的多样性和疾病的进展；2)了解前列腺素E_2和转化生长因子-�1调节DNA甲基转移酶(DNMT)和DNA甲基化模式的机制，以及它们如何导致成纤维细胞DNA甲基化和基因表达的差异。来自特发性肺纤维化和非纤维化肺的成纤维细胞将使用Illumina Human Metylation450微珠芯片阵列进行甲基化差异检测。差异甲基化基因将与差异基因表达和临床疾病的纵向进展相关。在目标2中，将在正常和特发性肺纤维化成纤维细胞中检测前列腺素E_2和转化生长因子-�_1对DNA甲基化和DNA甲基化机制的影响。这种方法是创新的，因为它将利用无偏见的“下一代”技术，将整个基因组DNA甲基化模式与来自IPF患者的纵向临床数据相关联，并描述调节DNA甲基化机制和特定DNA甲基化模式的机制。这个项目意义重大，因为它将建立DNA甲基化作为一种机制，有助于差异基因表达、成纤维细胞功能改变，并最终导致临床疾病进展，提供对如何调控糖尿病肾病甲基化模式的洞察，并确定可能成为未来治疗靶向的新途径。

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with no known effective therapy. Despite the dismal median survival, the prognosis of individual patients is variable and unpredictable, with some patients remaining stable and others deteriorating rapidly. The mechanisms that account for the variability in patients' clinical disease course are unknown. Fibroblasts are the main effector cell in fibrosis, and their relentless and exuberant activity is felt to contribute to the progressive nature of IPF. IPF fibroblasts exhibit gene expression changes that persist in culture even after multiple cell passages, suggesting that epigenetic modifications may be responsible for some of these gene expression differences. Alterations in DNA methylation of select genes have been identified in IPF fibroblasts, but a global assessment of the DNA methylomic abnormalities in IPF and their contribution to disease progression has never been assessed. The objectives of this project are to explore how genome-wide DNA methylomic and transcriptomic changes in IPF cells contribute to the clinical variability in IPF and determine how specific anti- and profibrotic mediators, prostaglandin E2 (PGE2) and transforming growth factor (TGF)-�1 respectively, contribute to altered DNA methylation patterns in IPF. The central hypothesis is that fibroblasts of IPF patients acquire distinct alterations in the DNA methylome in response to multiple extracellular signals, and that these distinct patterns of methylation contribute to both the heterogeneous gene expression profiles of fibroblasts and variable rates of clinical progression in IPF patients. This project has two specific aims: 1) examine how DNA methylomic changes in IPF fibroblasts contribute to both variable fibroblast gene expression patterns and clinical disease progression, and 2) understand the mechanisms by which PGE2 and TGF-�1 regulate DNA methyltransferases (DNMTs) and DNA methylation patterns and how they may contribute to differences in DNA methylation and gene expression of IPF fibroblasts. Fibroblasts from IPF and nonfibrotic lungs will be examined for methylation differences using the Illumina HumanMethylation450 Bead-Chip Array. Differentially methylated genes will be correlated with differential gene expression and longitudinal clinical disease progression. In Aim 2, the effects o PGE2 and TGF-�1 on DNA methylation and DNA methylation machinery will be examined in normal and IPF fibroblasts. The approach is innovative because it will utilize nonbiased, "next-generation" technologies to correlate whole genomic DNA methylation patterns with longitudinal clinical data from IPF patients and delineate mechanisms that regulate DNA methylation machinery and specific DNA methylation patterns. This project is significant because it will establish DNA methylation as a mechanism that contributes to differential gene expression, altered fibroblast function, and ultimately clinical disease progression, offer insight into how DN methylation patterns may be regulated, and identify novel pathways that may be candidates for future therapeutic targeting.