MicroRNA regulation of bone formation and repair

摘要 本研究的目的是确定两种非编码microRNA在调节细胞凋亡中的功能和机制。 在骨修复/疾病的情况下，体外和体内的骨生成。microRNAs（miRNAs）是一种小的， 编码靶向和抑制给定细胞类型内大量mRNA翻译的表观遗传调节因子， 导致许多途径和网络的调节。我们以前报道过差异表达的 在发育中的长骨的人胚胎生长板中的miRNAs，并鉴定了两种miRNAs（miR-miRNAs）。 181 a-1和miR-138），其在肥大软骨细胞中的表达高于在祖细胞中的表达。 软骨细胞，提示在调节软骨发生和/或软骨内骨化中的功能作用。我们 随后发现这些miRNAs在体外调控成骨中具有相反的作用：miR-181 a/B-1 （miR-181 a-1及其成簇的miRNA，miR-181 b-1）增强而miR-138抑制该过程。我们还发现 miR-181 a/B-1增强PI 3 K/AKT信号转导和线粒体代谢。新的初步数据显示 线粒体丙酮酸脱氢酶4（PDK 4）是miR-181 a/B-1的潜在靶基因。 PDK 4的功能是抑制丙酮酸脱氢酶复合物（PDC），导致线粒体膜电位降低。 新陈代谢.因此，miR-181 a/B-1对PDK 4的抑制可能部分解释了这种miRNA的增强作用 对线粒体呼吸和骨生成的影响。因此，在具体目标1中，我们计划测试miR-181 a/B- 1通过PI 3 K/AKT/FoxO 1通路直接和/或间接抑制PDK 4（假定FoxO 1是已知的PDK 4抑制剂）。 PDK 4转录激活因子）。我们还计划测试miR-138是否对PDK 4表达有相反的影响。 由于我们发现这种miRNA抑制PI 3 K/AKT信号传导以及与氧化应激相关的途径， 磷酸化这一目标还将探索两种PDK 4抑制剂药物对潜在增强 成骨利用异位骨化（HO）和骨折的小鼠模型，我们发现miR-138 过表达抑制HO形成和非软骨内骨折修复，而miR-181 a/B-1过表达抑制HO形成和非软骨内骨折修复。 表达增强软骨内骨折愈合。这些发现强调，在体外功能， 这两种miRNA在涉及新骨形成的修复/疾病模型中是体内可翻译的。因此，具体 目的2和3将研究体内调节miR-181 a/b-1或miR-138的作用，以试图抑制miR-181 a/b-1或miR-138的表达。 HO和促进骨折愈合。PDK 4抑制剂药物的作用也将在骨折中进行测试。 模型取决于目标1的发现。总的来说，这些研究旨在检验我们的总体假设， 体内适当靶向miR-181 a/B-1或miR-138将调节HO或骨修复期间的骨形成 这些作用部分是由于PI 3 K/AKT/FoxO 1/PDK 4信号转导和线粒体的调节， 新陈代谢.这些研究是重要的，因为新的机制信息将获得如何这些 miRNAs在骨生成过程中调节线粒体呼吸。此外， 将确定靶向每种miRNA作为治疗骨折或HO的手段。

ABSTRACT The goals of this study are to determine the function and mechanism of two non-coding microRNAs in regulating osteogenesis in vitro and also in vivo in the context of bone repair/disease. MicroRNAs (miRNAs) are small, non- coding epigenetic regulators that target and suppress translation of numerous mRNAs within a given cell type, resulting in modulation of many pathways and networks. We previously reported on differentially-expressed miRNAs within the human embryonic growth plate of developing long bones and identified two miRNAs (miR- 181a-1 and miR-138) that were more highly expressed in hypertrophic chondrocytes compared to progenitor chondrocytes, suggesting functional roles in regulating chondrogenesis and/or endochondral ossification. We subsequently found that these miRNAs have opposing roles in regulating osteogenesis in vitro: miR-181a/b-1 (miR-181a-1 and its clustered miRNA, miR-181b-1) enhances while miR-138 inhibits this process. We also found that miR-181a/b-1 enhances PI3K/AKT signaling and mitochondrial metabolism. New preliminary data suggests that the mitochondrial enzyme, pyruvate dehydrogenase 4 (PDK4) is a potential target gene of miR-181a/b-1. PDK4 functions to inhibit the pyruvate dehydrogenase complex (PDC) resulting in decreased mitochondrial metabolism. Thus, suppression of PDK4 by miR-181a/b-1 may partly explain the enhancing effects of this miRNA cluster on mitochondrial respiration and osteogenesis. In Specific Aim 1, we therefore plan to test if miR-181a/b- 1 suppresses PDK4 directly and/or indirectly via the PI3K/AKT/FoxO1 pathway (given that FoxO1 is a known transcriptional activator of PDK4). We also plan to test if miR-138 has opposing effects on PDK4 expression since we found that this miRNA suppresses PI3K/AKT signaling as well as pathways associated with oxidative phosphorylation. This aim will also explore the effects of two PDK4 inhibitor drugs on potentially enhancing osteogenesis. Utilizing murine models of heterotopic ossification (HO) and bone fracture, we found that miR-138 over-expression suppresses HO formation and non-endochondral bone fracture repair while miR-181a/b-1 over- expression enhances endochondral bone fracture healing. These findings highlight that the in vitro function of both miRNAs is translatable in vivo in repair/disease models involving new bone formation. Therefore, Specific Aims 2 and 3 will investigate the effects of modulating miR-181a/b-1 or miR-138 in vivo to attempt to suppress HO and enhance fracture healing, respectively. Effects of PDK4 inhibitor drugs will also be tested in the fracture models depending on findings from Aim 1. Overall, these studies are designed to test our overall hypothesis that appropriate targeting of miR-181a/b-1 or miR-138 in vivo will modulate bone formation during HO or bone repair and that these effects are due, in part, to regulation of PI3K/AKT/FoxO1/PDK4 signaling and mitochondrial metabolism. These studies are important because new mechanistic information will be gained on how these miRNAs regulate mitochondrial respiration during osteogenesis. In addition the potential therapeutic value of targeting each miRNA as a means to treat bone fractures or HO will be determined.