Role of miR29 in osteoclastogenesis

 描述（由申请方提供）：RANKL及其负调控因子OPG的相对水平对于破骨细胞生成的启动和进展至关重要。然而，调节破骨细胞生成的幅度和克里思的因素还不太清楚，越来越多的证据表明microRNAs（miRNAs，miRs）在这一过程中发挥重要作用。一种miRNA可以调节特定通路内的结构或信号分子家族;因此，除了调节信号通路之间的串扰之外，miRNA还可以放大或抑制细胞外信号的作用，平衡和缓冲细胞反应。 在破骨细胞谱系中，miR-29家族成员高度表达，在破骨细胞分化期间增加，并促进破骨细胞生成。我们的体外数据表明，miR-29促进破骨细胞生成，至少部分地，通过促进破骨细胞命运的承诺，并通过支持细胞迁移。为了解决可能的机制，我们确定了一组新的miR-29靶点，这些靶点具有调节定型、细胞骨架组织、细胞运动和破骨细胞功能的潜力。此外，我们开发了一种体内模型（miR-29竞争性抑制剂或“海绵”小鼠），用于研究miR-29在破骨细胞中的作用，这些小鼠显示骨小梁体积增加。 我们推测miR-29至少部分通过支持细胞迁移和谱系定型促进破骨细胞生成，因此对正常骨重建至关重要。在目标1中，我们将使用TRAP-miR-29海绵小鼠对miR-29在破骨细胞生成过程中的功能进行全面分析。我们将表征miR-29海绵小鼠的骨骼表型，并确定miR-29海绵对原代细胞谱系定型、分化、凋亡和再吸收的影响。活细胞成像将用于评价细胞运动性和融合的参数。在目标2中，我们将通过研究2个新验证的受RANKL强烈调控的miR-29靶点（SRGAP 2和CD 93）的功能来确定miR-29及其靶点控制破骨细胞生成的机制，这些靶点之前未在破骨细胞谱系中进行过研究。将在体外和体内进行敲低和过表达研究，并量化对定型、运动性、细胞骨架组织和再吸收的影响。 影响：了解miR-29及其靶点如何调节破骨细胞生成将为破骨细胞生成过程本身提供重要的新信息。此外，基于miRNA的疗法代表了治疗疾病的强大工具，并且正在临床试验中。限制其应用的一个严重因素是我们对多种细胞类型中的miRNA靶点和调控网络的知识存在差距。这些信息对于开发新的治疗方法、将其转化为临床以及预测疗效和预后至关重要。 安全为代价的我们在破骨细胞中研究的一些机制可能在其他细胞系统中也很活跃;因此这项工作也有助于我们理解miR-29家族在癌症、衰老和糖尿病中的作用。

 DESCRIPTION (provided by applicant): The relative levels of RANKL and its negative regulator, OPG, are critical for the initiation and progression of osteoclastogenesis. However, factors that modulate the amplitude and tempo of osteoclastogenesis are less well understood, and growing evidence indicates that microRNAs (miRNAs, miRs) play an important role in this process. One miRNA can regulate families of structural or signaling molecules within a particular pathway; thus miRNAs can amplify or dampen the effects of extracellular signals, balancing and buffering cellular responses, in addition to regulating the cross talk between signaling pathways. In the osteoclast lineage, miR-29 family members are highly expressed, are increased during osteoclast differentiation, and promote osteoclastogenesis. Our in vitro data demonstrate that miR-29 promotes osteoclastogenesis, at least in part, by promoting commitment to the osteoclast fate and by supporting cell migration. To address possible mechanisms, we identified a novel set of miR-29 targets with the potential to regulate commitment, cytoskeletal organization, cell motility and osteoclast function. In addition, we developed an in vivo model (miR-29 competitive inhibitor or "sponge" mice) for studying miR-29 actions in osteoclasts, and these mice display increased trabecular bone volume. We hypothesize that miR-29 promotes osteoclastogenesis, at least in part, by supporting cell migration and lineage commitment, and therefore is essential for normal bone remodeling. In Aim 1, we will perform a comprehensive analysis of miR-29 function during osteoclastogenesis using TRAP-miR-29 sponge mice. We will characterize the skeletal phenotype of miR-29 sponge mice, and determine effects of the miR-29 sponge on lineage commitment, differentiation, apoptosis and resorption in primary cells. Live cell imaging will be used to evaluate parameters of cell motility and fusion. In Aim 2, we will determine the mechanisms by which miR-29 and its targets control osteoclastogenesis, by studying the function of 2 newly validated miR-29 targets that are strongly regulated by RANKL, but have not been previously studied in the osteoclast lineage: SRGAP2 and CD93. Knock down and over expression studies will be performed in vitro and in vivo, and effects on commitment, motility, cytoskeletal organization and resorption will be quantified. Impact: Understanding how miR-29 and its targets regulate osteoclastogenesis will provide important new information about the process of osteoclastogenesis itself. Further, miRNA-based therapeutics represent powerful tools treating disease, and are in clinical trials. A serious limit to their utility is the gap in our knowledge o miRNA targets and regulated networks in multiple cell types. Such information is critical for the development of novel therapeutics, their translation to the clinic, and for predicting efficacy and safety. Some mechanisms that we study in osteoclasts could be active in other cell systems; therefore this work could also contribute to our understanding of the role of the miR-29 family in cancer, aging and diabetes.