Regulation of Skeletal Development by microRNAs

描述（由申请人提供）：该项目的长期目标是确定小的非编码microrna （mirna）在调节软骨发育和体内平衡中的功能作用。获得这些新知识对于产生基于微rna的替代策略来治疗创伤性软骨缺陷和防止骨关节炎（人类最常见的退行性疾病）的进展至关重要。为了阐明需要进一步研究哪些mirna，我们对从人胚胎软骨组织中提取的RNA进行了基于TaqMan(R)的微阵列，以鉴定体内前体、分化和肥大软骨细胞之间高表达的mirna以及差异表达的mirna。从这些研究中，我们已经生成了一个令人兴奋的候选mirna列表，这些候选mirna尚未在软骨生物学的背景下进行研究。我们假设这些mirna在调节软骨细胞分化的特定阶段和/或在病理生理条件下参与调节成熟软骨组织的稳态。在Specific Aim 1中，我们将利用体外人间充质干细胞（MSC）测定系统和体外小鼠胚胎跖骨外植体来确定mirna在调节软骨细胞分化阶段中的功能。一种最先进的免疫沉淀RNA诱导沉默复合物（RISCs）方法将应用于RNA测序，以鉴定miRNA靶基因和受影响的细胞途径。在Specific Aim 2中，将使用一个控制良好的非侵入性小鼠关节负荷模型来确定软骨细胞中特定mirna的表达水平如何在病理生理条件下发生改变。这些mirna在调节软骨细胞分解代谢或合成代谢途径中的功能也将被阐明。在临床上，该应用程序的研究是重要的，因为存在以下未满足的需求：1)使用干细胞修复或再生关节软骨的有效策略；2)治疗干预治疗

DESCRIPTION (provided by applicant): The long term goals of this project are to determine the functional roles of small, non-coding microRNAs (miRNAs) in regulating cartilage development and homeostasis. Acquiring such new knowledge will be critical for the generation of alternative microRNA-based strategies to treat trauma-induced cartilage defects and prevent progression toward osteoarthritis, the most common degenerative disease in humans. To elucidate which miRNAs to study further, we carried out TaqMan(R)-based microarrays on RNA extracted from human embryonic cartilage tissue to identify highly-expressed miRNAs as well as differentially- expressed miRNAs between precursor, differentiated and hypertrophic chondrocytes in vivo. From these studies, we have generated an exciting candidate list of miRNAs to pursue that has not yet been studied within the context of cartilage biology. We hypothesize that these miRNAs are functional in regulating specific phases of chondrocyte differentiation and/or are involved in modulating homeostasis of mature cartilage tissue under pathophysiological conditions. In Specific Aim 1, we will determine the function of miRNAs in regulating phases of chondrocyte differentiation by utilizing a human mesenchymal stem cell (MSC) assay system in vitro and murine embryonic metatarsal bone explants ex vivo. A state-of-the-art approach to immunoprecipitate RNA- induced silencing complexes (RISCs) followed by RNA sequencing will be applied to identify miRNA target genes and the cellular pathways affected. In Specific Aim 2, a well-controlled, non-invasive murine joint loading model will be used to determine how expression levels of specific miRNAs in chondrocytes are altered in response to pathophysiological conditions. The function of these miRNAs in regulating catabolic or anabolic pathways in chondrocytes will also be elucidated. Clinically, the research proposed in this application is important given that there is an unmet need for: 1) effective strategies to repair or regenerate articular cartilage using stem cells and 2) therapeutic interventions to treat injured articular cartilage, control homeostasis and prevent development of post-traumatic osteoarthritis. Since miRNAs can fine-tune the expression of multiple genes, they may be more useful tools than siRNA in complex diseases such as osteoarthritis that involve "interactomes" of molecular players. Notably, the studies proposed in this application are timely given the emergence of miRNAs as potential therapeutic targets in other disease scenarios in addition to current advances being made to deliver small molecules to mimic or inhibit miRNA function in vivo.