CDKN2B as a Novel Epigenetically Regulated Gene in Idiopathic Pulmonary Fibrosis

 描述（由申请方提供）：虽然特发性肺纤维化（IPF）的病因不明，可能涉及多种机制，但过度纤维增生仍然是IPF病理学的中心标志。在疾病发病机制中发挥关键作用的许多细胞类型中，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被多个研究者证明表现出促纤维化的体外表型;即使在多次细胞传代后培养细胞时也经常进行这些观察，表明表观遗传修饰可能是这些表型变化的原因。通过对IPF成纤维细胞中DNA甲基化模式的全球调查，发现CDKN 2B是IPF成纤维细胞中最差异甲基化的基因之一。尽管以前从未在IPF的背景下研究过，但已显示CDKN 2B在许多癌症中高度差异甲基化，并调节肿瘤中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CDKN 2B表达减少导致IPF细胞活化表型的机制，并了解其表达如何受到DNA甲基化的调节，以及甲基化如何受到长链非编码RNA（lncRNA）的表达和作用的影响。我们的中心假设是，由lncRNA CDKN 2B-反义（AS）1驱动的CDKN 2B超甲基化导致IPF成纤维细胞中CDKN 2B表达减少，从而导致肌成纤维细胞分化和凋亡抗性增加。该项目有三个具体目标：1）确定CDKN 2B的表达减少如何促成IPF成纤维细胞的促纤维化表型; 2）确定DNA甲基化如何调节CDKN 2B的表达; 3）阐明CDKN 2B-AS 1促成IPF成纤维细胞中CDKN 2B的DNA超甲基化的机制。将通过检查来自正常肺和IPF患者的原代成纤维细胞以及通过小鼠建模实验来探索CDKN 2B在肺纤维化中的生物学作用。将进行DNA甲基化分析，并检查lncRNA在调节CDKN 2B DNA甲基化中的作用。该项目具有创新性，因为它试图了解可能对纤维增生至关重要的基因的功能以及表观遗传调控，但以前从未在IPF中进行过研究。这些研究的完成预计将确定CDKN 2B对IPF成纤维细胞生物学的生物学重要性，并阐明调节其表达的机制，并有助于如何建立差异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，而且还促进了我们对DNA甲基化变化如何产生并导致IPF基因表达差异的理解。

 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.