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、非编码的microRNAs(MiRNAs)在调节软骨发育和动态平衡中的功能作用。获得这些新知识对于产生基于microRNA的替代策略至关重要，这些策略可以治疗创伤引起的软骨缺陷，并防止进展为骨关节炎，骨关节炎是人类最常见的退行性疾病。为了阐明哪些miRNAs需要进一步研究，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，以识别体内前体、分化和肥大软骨细胞中高表达和差异表达的miRNAs。从这些研究中，我们产生了一个令人兴奋的候选miRNAs列表，这些miRNAs尚未在软骨生物学的背景下进行研究。我们推测这些miRNAs在调控软骨细胞分化的特定阶段和/或参与调节成熟软骨组织在病理生理条件下的动态平衡。在具体目标1中，我们将利用体外培养的人骨髓间充质干细胞(MSC)检测系统和小鼠胚胎跖骨组织块，研究miRNAs在软骨细胞分化过程中的调控作用。一种先进的免疫沉淀RNA诱导沉默复合体(RISCs)的方法将被应用于识别miRNA靶基因和受影响的细胞通路。在特定目标2中，将使用一个控制良好的、非侵入性的小鼠关节负荷模型来确定软骨细胞中特定miRNAs的表达水平如何随着病理生理条件的变化而改变。这些miRNAs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，本申请中提出的研究是重要的，因为在以下方面的需求尚未得到满足：1)使用干细胞修复或再生关节软骨的有效策略；2)治疗干预措施。 损伤关节软骨，控制动态平衡，防止创伤后骨关节炎的发展。由于miRNAs可以微调多个基因的表达，它们可能是比siRNA更有用的工具，用于治疗涉及分子参与者相互作用的复杂疾病，如骨关节炎。值得注意的是，除了目前在运送小分子以模拟或抑制体内miRNA功能方面取得的进展外，鉴于miRNAs在其他疾病场景中作为潜在治疗靶点的出现，本申请中提出的研究是及时的。

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.