The role of microRNAs in chondrogenesis and arthritis

描述（由申请人提供）：发育中骨骼框架的精确模式依赖于对软骨形成的适当控制，软骨形成是一个多步骤过程，在此过程中，间充质细胞分化为软骨细胞。关节软骨破坏是关节疾病（包括骨关节炎（OA）和类风湿性关节炎（RA））发病机制的核心因素。对软骨形成的分子机制的研究具有发育生物学意义，并有可能导致组织工程和新型关节炎治疗的新方法。microRNAs（miRNAs）是一类由约22个核苷酸组成的非编码RNA家族，通过转录后机制调控基因表达。迄今为止，软骨细胞特异性miRNAs，其上游分子信号和靶基因在很大程度上是未知的。在我们的初步实验中，我们进行了软骨细胞中miRNA表达和miR-140表达的微阵列分析，在胚胎发育和成人软骨软骨细胞中的软骨发育。这些研究表明，miR-140具有最多的软骨特异性表达模式。Carcinoma特异性Sox 9敲除小鼠不表达miR-140，ChIP分析和EMSA显示与前体miR-140接近的潜在Sox 9结合区与Sox 9相关，表明Sox 9是miR-140基因表达的关键调节因子。与正常软骨相比，通过IL-1？刺激软骨细胞，骨关节炎软骨中的miR-140表达下调。miR-140敲除小鼠在6个月时膝关节中的骨关节炎变化较早发作。微阵列和生物信息学分析揭示了软骨细胞中一组可能的miR-140靶基因，包括ADAMTS-5，这是骨关节炎中的关键组织降解酶。这些观察结果支持以下假设：miR-140是软骨发育和稳态的新型调节剂，并且其表达和功能的那些变化在影响关节软骨的疾病（例如骨关节炎）中起重要作用。 我们提出以下具体目的：目的1：研究Sox 9对骨髓间充质干细胞和软骨细胞中miR-140表达的转录调控。目的2：检测miR-140在软骨发生和软骨发育中的作用。目的3：探讨miR-140在骨关节炎中的表达及功能。目的4：确定软骨细胞和间充质干细胞中miR-140的新靶基因。 拟议的研究有可能揭示控制软骨发育和稳态的重要的新调控途径，并为疾病机制和治疗干预提供新的见解。 公共卫生相关性：尽管最近在理解OA发病机制方面取得了实质性进展，但目前还没有针对最常见关节疾病的疾病改善疗法。研究miR-140在MSC和软骨细胞中的作用可能为软骨细胞生物学提供新的见解，并为骨关节炎的组织工程和治疗策略提供新方法的基础。

DESCRIPTION (provided by applicant): The precise patterning of the developing skeletal framework relies on the appropriate control of chondrogenesis, a multistep process during which mesenchymal cells differentiate into chondrocytes. Articular cartilage destruction is a central factor in the pathogenesis of joint diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). Research on the molecular mechanisms of chondrogenesis is of developmental biological interest and has the potential to lead to novel approaches in tissue engineering and novel arthritis treatments. MicroRNAs (miRNAs) are a family of ~22-nucleotide (nt) noncoding RNAs that regulate gene expression by posttranscriptional mechanisms. To date, chondrocyte specific miRNAs, their upstream molecular signals, and target genes are largely unknown. In our preliminary experiments, we performed microarray profiling of miRNA expression in chondrocytes and miR-140 expression in cartilage development during embryogenesis and adult cartilage chondrocytes. These studies demonstrated that miR-140 has the most cartilage specific expression pattern. Cartilage specific Sox9 null mice did not express miR-140 and the potential Sox9 binding region close to pre-miR-140 was associated with Sox9 by ChIP assay and EMSA, suggesting that Sox9 is a key regulator of miR-140 gene expression. miR-140 expression was down-regulated in osteoarthritic cartilage compared with normal cartilage and by IL-1¿ stimulation of chondrocytes. miR-140 null mice showed earlier onset of osteoarthritic changes in knee joint at 6 months. Microarray and bioinformatics analysis revealed a set of possible miR-140 target genes in chondrocytes, including ADAMTS-5, which is a key tissue-degrading enzyme in osteoarthritis. These observations support the hypothesis that miR-140 is a novel regulator of cartilage development and homeostasis and those changes in its expression and function play an important role in diseases affecting articular cartilage, such as osteoarthritis. We propose the following specific aims: Aim 1: Investigate the transcriptional regulation of miR-140 expression by Sox9 in mesenchymal stem cells and chondrocytes. Aim 2: Examine the role of miR-140 in chondrogenesis and cartilage development. Aim 3: Determine the expression and function of miR-140 in osteoarthritis. Aim 4: Identify novel target genes of miR-140 in chondrocytes and mesenchymal stem cells. The proposed studies have the potential to reveal important new regulatory pathways that control cartilage development and homeostasis and open new insight on disease mechanisms and therapeutic interventions. PUBLIC HEALTH RELEVANCE: Despite substantial recent progress in understanding OA pathogenesis, disease-modifying therapies for the most prevalent joint disease are not available. Examining the role of miR-140 in MSC and chondrocytes may provide new insight into cartilage cell biology as well as a basis for novel approaches in tissue engineering and therapeutic strategies for osteoarthritis.