MicroRNA regulation of bone formation and repair

摘要 本研究的目的是确定两个非编码的microRNAs在调控中的功能和机制 在骨修复/疾病的背景下，体外和体内的成骨。MicroRNAs(MiRNAs)是小的、非 编码针对并抑制给定细胞类型内大量mRNA的翻译的表观遗传调节因子， 从而导致许多路径和网络的调制。我们之前曾报道过差异表达 在发育中的人类长骨的胚胎生长板中发现了两个miRNAs(miR- 181a-1和miR-138)在肥大软骨细胞中的表达高于祖细胞 软骨细胞，提示在调节软骨形成和/或软骨内成骨中起功能作用。我们 随后发现，这些miRNAs在调节体外成骨方面具有相反的作用：miR-181a/b-1 (miR-181a-1及其簇miRNA，miR-181b-1)促进这一过程，而miR-138抑制这一过程。我们还发现 MiR-181a/b-1增强PI3K/AKT信号转导和线粒体代谢。新的初步数据表明 线粒体酶丙酮酸脱氢酶4(PDK4)是miR-181a/b-1的潜在靶基因。 PDK4抑制丙酮酸脱氢酶复合体(PDC)，导致线粒体减少 新陈代谢。因此，miR-181a/b-1对PDK4的抑制可能部分解释了该miRNA的增强作用 聚集在线粒体呼吸和成骨上。因此，在具体目标1中，我们计划测试miR-181a/b- 1通过PI3K/AKT/FoxO1途径直接和/或间接抑制PDK4(鉴于FoxO1是一种已知的 PDK4转录激活子)。我们还计划测试miR-138是否对PDK4的表达具有相反的影响 由于我们发现这种miRNA抑制PI3K/AKT信号以及与氧化相关的通路 磷酸化。这一目标还将探索两种PDK4抑制剂药物对潜在的增强 成骨作用。利用小鼠异位骨化(HO)和骨折模型，我们发现miR-138 MiR-181a/b-1过表达抑制HO形成和非软骨内骨折修复 表达促进软骨内骨骨折愈合。这些发现突显了细胞的体外功能 在涉及新骨形成的修复/疾病模型中，这两种miRNAs都是可在体内翻译的。因此，具体来说 AIMS 2和3将研究在体内调节miR-181a/b-1或miR-138的效果，试图抑制 HO和促进骨折愈合。PDK4抑制剂药物的效果也将在骨折中进行测试 模型取决于目标1的发现。总体而言，这些研究旨在测试我们的总体假设 在体内适当靶向miR-181a/b-1或miR-138将调节HO或骨修复期间的骨形成 这些效应部分归因于对PI3K/AKT/FoxO1/PDK4信号和线粒体的调节 新陈代谢。这些研究很重要，因为将获得新的机械信息，说明这些 MiRNAs在成骨过程中调节线粒体的呼吸。此外，还具有潜在的治疗价值 以每个miRNA为靶点作为治疗骨折或HO的手段将被确定。

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.