The Role of miR-219a-5p in Bone Metabolism

项目总结/摘要 骨质疏松症是一种常见的临床疾病，其特征是骨量低，增加了脆性的风险 老年人骨折。由于骨质疏松症患者的骨形成明显受损， 对调节骨代谢的基本分子机制的完全理解可能 导致新疗法的发展。因此，鉴定新的分子途径， 影响骨形成对于对抗骨质疏松症的临床治疗的发展至关重要。我们 以前的工作已经明确地确定了抗成骨转录因子，视黄酸受体- 相关孤儿受体β（Rorβ）在成骨细胞分化中起抑制作用。增加表达 生理老化过程中Rorβ的水平有助于骨丢失，因为缺乏Rorβ的小鼠表现出更高的骨密度。 通过激活骨合成代谢Wnt途径。这导致了这样一种观点，即抑制Rorβ可能 代表了在整个衰老过程中增加骨量的新范例。我们假设 microRNAs（miRs）可能控制Rorβ水平，并发现一种特异性miR，miR-219 a-5 p（miR-219 a）， 预测的Rorβ-调节miR，表现出与Rorβ相反的表达模式，表明可能的 骨中的Rorβ/miR-219 a调节轴。我们证明，miR-219 a直接调节Rorβ水平， miR-219 a模拟物的递送增强了成骨细胞的分化，并且miR-219 a 拮抗剂抑制分化。我们进一步表明，miR-219 a增加了骨合成代谢的活性， Wnt途径。因此，我们认为miR-219 a具有骨合成代谢作用，可用于保护骨量 随着年龄的增长，可能通过增加Wnt途径活性。使用一种新的小鼠模型，我们可以激活 miR-219 a以组织特异性方式表达，在目的1中，我们将确定miR-219 a在Rorβ-受体中的作用。 Rorβ表达的细胞在成年骨块上或骨折后，假设Rorβ的下调 通过miR-219 a将增加骨量并加速骨折愈合。在目标2中，我们将探索更多 miR-219 a通过激活各种骨细胞谱系中的miR-219 a在骨稳态中的一般作用， 假设这些谱系中Rorβ和/或其他miR-219 a靶点的下调将增加骨密度， 质量随着年龄的增长。最后，在目标3中，我们将进行体外实验以探索 miR-219 a如何调节Wnt通路，并确定miR-219 a直接靶向Rorβ是否影响 Wnt活性与骨细胞功能。完成这些关键研究不仅可以提供更完整的 了解miR-219 a在骨中的功能，但如果是阳性的，也将提供一个强有力的理由， miR-219 a模拟物作为一种新的治疗策略在各种临床条件下刺激骨形成。

PROJECT SUMMARY/ABSTRACT Osteoporosis is a common clinical condition characterized by low bone mass that increases the risk of fragility fractures in the elderly population. Since bone formation is clearly impaired in osteoporotic patients, a more complete understanding of the fundamental molecular mechanisms that regulate bone metabolism is likely to lead to the development of novel therapies. Therefore, identification of novel molecular pathways which influence bone formation is crucial to the development of clinical treatments to combat osteoporosis. Our previous work has definitively established that the anti-osteogenic transcription factor, retinoic acid receptor- related orphan receptor-beta (Rorβ) plays an inhibitory role in osteoblast differentiation. Increasing expression levels of Rorβ during physiological aging contributes to bone loss, as mice lacking Rorβ exhibit higher bone mass through activation of the bone anabolic Wnt pathway. This leads to the notion that inhibition of Rorβ may represent a novel paradigm to increase bone mass throughout the aging process. We hypothesized that microRNAs (miRs) may control Rorβ levels, and found that a specific miR, miR-219a-5p (miR-219a), the top predicted Rorβ-regulatory miR, exhibits an inverse expression pattern to Rorβ, suggesting a potential Rorβ/miR-219a regulatory axis in bone. We demonstrate that miR-219a directly regulates Rorβ levels in osteoblasts, that delivery of miR-219a mimics enhances osteoblast differentiation, and that miR-219a antagonists suppress differentiation. We further show that miR-219a increases the activity of the bone anabolic Wnt pathway. Therefore, we propose that miR-219a is bone anabolic and may be used to preserve bone mass with age, possibly through increasing Wnt pathway activity. Using a novel mouse model where we can activate miR-219a expression in a tissue-specific manner, in Aim 1 we will determine the effects of miR-219a in Rorβ- expressing cells on adult bone mass or following a fracture, with the hypothesis that downregulation of Rorβ through miR-219a will increase bone mass and accelerate fracture healing. In Aim 2, we will explore the more general effects of miR-219a in bone homeostasis by activating miR-219a in various bone cell lineages, with the hypothesis that downregulation of Rorβ, and/or other miR-219a targets in these lineages, will increase bone mass with age. Finally, in Aim 3 we will perform in vitro experiments to explore the molecular mechanism of how miR-219a regulates the Wnt pathway and determine whether direct targeting of Rorβ by miR-219a affects Wnt activity and bone cell function. Completion of these pivotal studies will not only provide a more complete understanding of miR-219a function in bone, but if positive will also provide a strong justification for pursuing miR-219a mimics as a novel therapeutic strategy to stimulate bone formation in various clinical conditions.