The role of microRNAs in chondrogenesis and arthritis

描述(申请人提供)：发育中的骨骼框架的精确模式依赖于对软骨形成的适当控制，在此过程中，间充质细胞分化为软骨细胞的多步骤过程。关节软骨破坏是关节疾病发病的中心因素，包括骨性关节炎(OA)和类风湿关节炎(RA)。研究软骨形成的分子机制具有发展生物学意义，并有可能导致组织工程和新型关节炎治疗的新方法。MicroRNAs(MiRNAs)是一个由~22个核苷酸(NT)组成的非编码RNA家族，通过转录后机制调节基因表达。到目前为止，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，我们对胚胎发育过程中软骨细胞和成年软骨细胞中miRNA的表达和miR-140在软骨发育中的表达进行了微阵列分析。这些研究表明miR-140具有最具软骨特异性的表达模式。软骨特异的Sox9缺失小鼠不表达miR-140，芯片分析和EMSA显示靠近miR-140前蛋白的潜在Sox9结合区与Sox9相关，提示Sox9是miR-140基因表达的关键调控因子。MIR-140在骨性关节炎软骨中的表达较正常软骨下调，并受IL-1β刺激的软骨细胞的影响。MIR-140空白小鼠在6个月时表现出较早的膝关节骨关节炎改变。基因芯片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，它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是一种新的软骨发育和动态平衡调节因子，其表达和功能的变化在影响关节软骨的疾病中发挥着重要作用，如骨关节炎。目的1：研究Sox9对间充质干细胞和软骨细胞miR-140表达的转录调控作用。目的2：研究miR-140在软骨发生和软骨发育中的作用。目的3：探讨miR-140在骨性关节炎中的表达及功能。目的4：在软骨细胞和间充质干细胞中寻找miR-140的新靶基因。拟议的研究有可能揭示控制软骨发育和动态平衡的重要新调控途径，并为疾病机制和治疗干预开辟新的视角。 公共卫生相关性：尽管最近在了解骨性关节炎发病机制方面取得了实质性进展，但最常见的关节疾病的疾病修正疗法尚不存在。检测miR-140在MSC和软骨细胞中的作用可能会为软骨细胞生物学提供新的见解，并为组织工程和骨关节炎的治疗策略提供新的方法基础。

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.