The Role of MicroRNA in Osteoarthritis

摘要 近年来骨关节炎（OA）研究取得了重大进展，包括产生 小鼠模型，包括遗传和手术诱导的 OA 小鼠模型，显示出 OA 样表型。 这些 OA 小鼠模型的共同特征之一是 Runx2 的上调。因为 Runx2 是 直接调节基质降解酶编码基因表达的关键转录因子， 例如Mmp9、Mmp13和Adamts5，这种上调会导致这些基因的表达增加。 关键问题已经提出来了。 1) Runx2 是介导关节组织中 OA 发展的中心分子吗？ 2) 操纵Runx2表达可以用于治疗OA疾病吗？ miRNA 是内源性非 编码RNA并在RNA稳定性和蛋白质表达的调节中发挥重要作用。之前的研究 我们实验室的研究表明，两个同源 miRNA miR-204/-211 可以结合 Runx2 并调节 Runx2 间充质干细胞（MSC）中的表达。确定这些 miRNA 在 Runx2 中的功能 在 OA 开发中，我们最近创建了 miR-204 和 miR-211 floxed 等位基因，并进一步 通过繁殖 miR-204flox/flox 和 miR- 产生 miR-204/-211 双 KO 小鼠（dmiRPrx1 和 dmiRAgc1ER） 带有 Prx1-Cre（针对肢体 MSC）或 Agc1-CreER（针对成人关节软骨细胞）的 211flox/flox 小鼠 转基因小鼠删除这些 miRNA。在 dmiRPrx1 和 dmiRPrx1 中观察到严重的 OA 样表型 dmiRAgc1ER KO 小鼠和整个膝关节组织均受到 miR-204/-211 缺失的影响，包括严重丢失 关节软骨、软骨下硬化、软骨赘/骨赘形成和滑膜增生。 我们的研究结果表明 miR-204 和 miR-211 是维持关节组织稳态的关键调节因子 可以作为 OA 治疗的关键靶点。该项目的基本假设是 miR- 204 和 miR-211 是 Runx2 表达的关键调节因子，在维持关节组织中发挥关键作用 体内平衡。已经提出了两个具体目标来检验这一假设。在目标1中，我们将充分 描述 dmiRAgc1ER KO 小鼠中 miR-204/-211 缺失对 OA 发展的纵向影响 确定软骨细胞中 Runx2 的缺失是否会逆转 dmiRAgc1ER KO 小鼠中观察到的 OA 表型。在 目标 2，我们将研究 OA 样本中 miR-204/-211 表达的变化，并确定体内是否存在 在手术诱发的 OA 中，给予 miR-204 将预防 OA 发展或减缓 OA 进展 鼠标模型。这些拟议的研究将为 OA 发展和机制提供新的见解。 将有助于制定 OA 治疗新策略。

Abstract Significant progress has been made in recent years in osteoarthritis (OA) research, including the generation of mouse models, including genetic and surgically-induced OA mouse models that show an OA-like phenotype. One feature that is common to these mouse models of OA is the up-regulation of Runx2. Because Runx2 is a key transcription factor directly regulating the expression of genes encoding for matrix degradation enzymes, such as Mmp9, Mmp13 and Adamts5, this up-regulation leads to the increase in expression of these genes. The key questions have been raised. 1) Is Runx2 a central molecule mediating OA development in joint tissue? 2) Could manipulation of Runx2 expression be used to treat OA disease? miRNAs are endogenous non- coding RNAs and play important roles in regulation of RNA stability and protein expression. Previous studies by our lab have shown that miR-204/-211, two homologous miRNAs, bind Runx2 and regulate Runx2 expression in mesenchymal stem cells (MSCs). To determine the function of these miRNAs in Runx2 regulation and in OA development, we have recently created miR-204 and miR-211 floxed alleles and further generated miR-204/-211 double KO mice (dmiRPrx1 and dmiRAgc1ER) by breeding miR-204flox/flox and miR- 211flox/flox mice with Prx1-Cre (targeting limb MSCs) or Agc1-CreER (targeting adult articular chondrocytes) transgenic mice to delete these miRNAs. A severe OA-like phenotype was observed in dmiRPrx1 and dmiRAgc1ER KO mice and entire knee joint tissues were affected by miR-204/-211 deletion, including severe loss of articular cartilage, subchondral sclerosis, chondrophyte/osteophyte formation, and synovial hyperplasia. Our findings suggest that miR-204 and miR-211 are key regulators in maintaining joint tissue homeostasis and could be served as critical targets for OA treatment. The underlying hypothesis of this project is that miR- 204 and miR-211 are key regulators of Runx2 expression and play critical roles in maintaining joint tissue homeostasis. Two specific aims have been proposed to test this hypothesis. In Aim 1, we will fully characterize the longitudinal effects of miR-204/-211 deletion on OA development in dmiRAgc1ER KO mice and determine if deletion of Runx2 in chondrocytes will reverse OA phenotype observed in dmiRAgc1ER KO mice. In Aim 2, we will investigate changes in miR-204/-211 expression in OA samples and determine if in vivo administration of miR-204 will prevent OA development or decelerate OA progression in surgically-induced OA mouse model. These proposed studies will provide novel insights into mechanisms of OA development and will help the development of new strategies for OA treatment.