CDKN2B as a Novel Epigenetically Regulated Gene in Idiopathic Pulmonary Fibrosis

 描述(申请人提供)：尽管特发性肺纤维化(IPF)的病因尚不清楚，可能涉及多种机制，但过度的纤维增殖仍然是IPF病理的主要特征。在疾病发病机制中起关键作用的多种细胞类型中，成纤维细胞最终是纤维化的效应细胞。来自IPF患者的成纤维细胞已经被多个研究人员证明在体外表现出促纤维化的表型；这些观察经常在细胞经过多次传代后培养时出现，这表明表观遗传修饰可能是导致这些表型变化的原因。通过对IPF成纤维细胞DNA甲基化模式的全球调查，CDKN2B被发现是IPF成纤维细胞中差异最大的甲基化基因之一。尽管以前从未在IPF的背景下进行过研究，但CDKN2B已被证明在许多癌症中存在高度分化的甲基化，并调节肿瘤的细胞增殖和细胞周期进展。我们的目标是确定高甲基化和CDKN2B表达下调导致IPF细胞激活表型的机制，并了解其表达如何受到DNA甲基化的调节，以及甲基化可能如何由长非编码RNA(LncRNA)的表达和作用决定。我们的中心假设是CDKN2B的高甲基化，在lncRNA CDKN2B反义(AS)1的驱动下，导致IPF成纤维细胞中CDKN2B的表达减少，从而导致肌成纤维细胞分化和抗凋亡能力增强。本项目有三个具体的目标：1)确定CDKN2B的表达降低如何导致IPF成纤维细胞的纤维化表型；2)确定DNA甲基化如何调控CDKN2B的表达；3)阐明CDKN2B-AS1在IPF成纤维细胞中促进CDKN2B DNA高甲基化的机制。通过对正常肺和IPF患者的原代成纤维细胞的检测和小鼠模型实验，探讨CDKN2B在肺纤维化中的生物学作用。将进行DNA甲基化分析，并研究lncRNAs在调节CDKN2B DNA甲基化中的作用。这个项目具有创新性，因为它试图了解一种可能对纤维增殖至关重要的基因的功能，以及表观遗传调节，但以前从未在IPF中进行过研究。这些研究的完成有望确定CDKN2B对IPF成纤维细胞生物学的生物学意义，并阐明其表达调控机制，并有助于建立差异DNA甲基化模式。这些发现不仅为未来的治疗提供了一个新的基因靶点，而且有助于我们理解DNA甲基化变化是如何发生的，并有助于IPF中基因表达的差异。

 DESCRIPTION (provided by applicant): Although the etiology of idiopathic pulmonary fibrosis (IPF) is unknown and may involve diverse mechanisms, excessive fibroproliferation remains a central hallmark in IPF pathology. Among the many cell types that play critical roles in disease pathogenesis, fibroblasts are ultimately the effector cell of fibrosis. Fibroblasts from IPF patiens have been shown by multiple investigators to exhibit a profibrotic in vitro phenotype; these observations were often made even when cells were cultured after multiple cell passages, suggesting that epigenetic modifications may be responsible for these phenotypic changes. Through a global survey of DNA methylation patterns in IPF fibroblasts, CDKN2B was discovered to be one of the most differentially methylated genes in IPF fibroblasts. Although never previously investigated in the context of IPF, CDKN2B has been shown to be highly differentially methylated in many cancers and to regulate cell proliferation and cell cycle progression in tumors. Our objectives are to determine the mechanism by which hypermethylation and diminished CDKN2B expression contributes to the activated phenotype of IPF cells, and to understand how its expression is regulated by DNA methylation, and how methylation may be dictated by the expression and actions of a long noncoding RNA (lncRNA). Our central hypothesis is that hypermethylation of CDKN2B, driven by the lncRNA CDKN2B-antisense (AS) 1, contributes to the diminished expression of CDKN2B in IPF fibroblasts, which leads to increased myofibroblast differentiation and apoptosis resistance. This project has three specific aims: 1) Determine how diminished expression of CDKN2B contributes to a profibrotic phenotype in IPF fibroblasts; 2) Determine how DNA methylation modulates the expression of CDKN2B; and 3) Elucidate the mechanisms by which CDKN2B-AS1 contributes to the DNA hypermethylation of CDKN2B in IPF fibroblasts. The biological actions of CDKN2B in pulmonary fibrosis will be explored by examining primary fibroblasts from normal lung and patients with IPF and by mouse modeling experiments. DNA methylation analysis will be performed, and the role of lncRNAs in regulating the DNA methylation of CDKN2B will be examined. This project is innovative because it seeks to understand the function, and additionally the epigenetic regulation, of a gene that may be critical to fibroproliferation, but has never been previously investigated in IPF. Completion of these studies is expected to establish the biological importance of CDKN2B to IPF fibroblast biology, and elucidate mechanisms that regulate its expression and contribute to how differential DNA methylation patterns are established. These findings will provide not only a novel gene target for future therapy, but also advance our understanding of how DNA methylation changes arise and contribute to gene expression differences in IPF.