Integrative Omics analysis of human cartilage in aging and osteoarthritis

摘要 骨性关节炎是最常见的关节疾病。尽管已经确定了许多药物靶点， 在临床前研究中取得成功，到目前为止，关于疾病修饰骨性关节炎药物的临床试验都失败了， 疼痛管理仍然不令人满意。测试疾病相关差异的方法 在临床前模型中，表达选定的基因或蛋白质并分析它们的功能已经产生了大量的 在OA中异常的通路和分子的数量。这些方法的局限性是：(I) 它们仅提供了对骨关节炎分子变化的选择性观察，以及(Ii)还没有成功的努力 将这些调查结果纳入网络，并根据目标作为开放式获取进程驱动力的相关性来确定目标的优先顺序。 该项目利用(I)我们从世界各地的捐赠者那里获得人类膝关节组织的机会和专业知识 整个成人年龄谱和骨性关节炎发育的所有阶段；(2)现存的和正在生长的人类膝关节组织 文库；(3)全基因组转录变化分析的技术进展，这提供了 公正和全面地看待软骨内稳态和骨关节炎的遗传图景；(Iv)我们的 在为多OMICS数据集的综合网络分析开发管道方面的专业知识。 我们的假设是，异常转录因子(TF)是异常基因的主要决定因素 驱动骨性关节炎发病机制的表达模式。我们的方法是从基因表达中产生数据 (MRNAseq)和增强子激活分析(gro-seq)，更准确地说是在单个 细胞水平，以确定软骨内稳态和骨关节炎的新信号、途径和关键调节因子。 目的1.正常人和骨性关节炎患者关节软骨单细胞水平的转录图谱。我们会 应用单细胞RNAseq技术鉴定正常人和骨性关节炎患者关节软骨细胞亚群 软骨。 目的2.增强子谱分析以确定骨关节炎致病基因表达模式的驱动因素。活性促进剂 其特征是存在增强子RNA(ERNAs)。我们将使用Gro-Seq来分析Erna 正常软骨细胞和骨关节炎软骨细胞的转录。这些结果将揭示路径和网络 在骨性关节炎中受到干扰，并确定骨性关节炎发病的主要调节因素。 目的3.验证：确认关节组织中TF表达和激活的差异，并分析其功能 关节组织细胞。我们将评估TF蛋白在软骨和其他关节中的表达和活性的差异。 并确定候选转录因子在调节骨关节炎相关基因模式表达中的作用。 影响：据我们所知，这是第一个检查全基因组mRNA表达谱的项目 在组织和单细胞水平上的健康和受OA影响的膝关节软骨，并将这一转录数据与 分析组织因子的表达和活性。该研究有可能发现新的途径和原理 分子开关作为治疗靶点。最终，这可能导致延迟或治疗骨性关节炎的干预措施。

ABSTRACT Osteoarthritis (OA) is the most prevalent joint disease. Although many drug targets have been identified that were successful in preclinical studies, clinical trials on disease modifying OA drugs thus far have failed and pain management remains unsatisfactory. Approaches such as testing disease-related differences in expression of selected genes or proteins and analyzing their function in preclinical models has yielded a large number of pathways and molecules that are abnormal in OA. The limitations of these approaches are (i) that they provide only a selective view of molecular changes in OA and (ii) there has been no successful effort in integrating these findings into networks and prioritizing targets by their relevance as drivers of the OA process. This project leverages (i) our access to and expertise in working with human knee tissues from donors across the entire adult age spectrum and at all stages of OA development; (ii) existing and growing human knee tissue libraries; (iii) technical advances in genome wide analyses of transcriptomic changes, which provide an unbiased and comprehensive view of the genetic landscape of cartilage homeostasis and OA; (iv) our expertise in developing pipelines for integrative network analysis of multi-Omics data sets. Our hypothesis is that dysregulation transcription factors (TFs) is a major determinant of the abnormal gene expression pattern that drives OA pathogenesis. Our approach is to generate data from gene expression (mRNAseq) and enhancer activation analysis (GRO-seq) at the tissue level and more precisely at the single cell level to identify novel signatures, pathways and key regulators of cartilage homeostasis and OA. Aim 1. The transcriptomic landscape of normal and OA human articular cartilage single cell levels. We will perform single-cell RNAseq to identify chondrocyte subpopulations in normal and OA human articular cartilage. Aim 2. Enhancer profiling to identify drivers of pathogenic gene expression patterns in OA. Active enhancers are characterized by the presence of enhancer RNAs (eRNAs). We will use GRO-seq to assay eRNA transcription in normal and OA chondrocytes. These results will reveal pathways and networks that are disrupted in OA and identify principal regulators of OA pathogenesis. Aim 3. Validation: Confirm differences in TF expression and activation in joint tissues and analyze function in joint tissue cells. We will assess differences in TF protein expression and activity in cartilage and other joint tissues and determine the role of candidate TFs in mediating expression of OA-associated gene patterns. Impact: To our knowledge, this is the first project to examine genome-wide mRNA expression profiles in healthy and OA-affected knee cartilage at tissue and single cell levels and linking this transcriptomic data with analysis of TF expression and activity. The study has potential discover novel pathways and principal molecular switches as therapeutic targets. Ultimately this may lead to interventions to delay or treat OA.