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，包括骨关节炎（OA）的产生。 小鼠模型，包括显示出OA样表型的遗传和药物诱导的OA小鼠模型。 这些OA小鼠模型的一个共同特征是Runx 2的上调。因为Runx 2是 直接调节编码基质降解酶的基因表达的关键转录因子， 如Mmp 9、Mmp 13和Adamts 5，这种上调导致这些基因的表达增加。 关键问题已经提出。1)Runx 2是关节组织中介导OA发展的中心分子吗？ 2)操纵Runx 2表达可以用于治疗OA疾病吗？miRNAs是内源性非- 在RNA稳定性和蛋白质表达的调控中起重要作用。以前的研究 我们实验室的研究表明，miR-204/-211，两种同源的miRNAs，结合Runx 2并调节Runx 2， 在间充质干细胞（MSC）中的表达。为了确定这些miRNAs在Runx 2中的功能， 调控和OA发展中，我们最近创建了miR-204和miR-211 floxed等位基因，并进一步 通过将miR-204 flox/flox和miR-211双基因敲除小鼠（dmiRPrx 1和dmiRAgc 1 ER） 具有Prx 1-Cre（靶向肢体MSC）或Agc 1-CreER（靶向成人关节软骨细胞）的211 flox/flox小鼠 转基因小鼠来删除这些miRNAs。在dmiRPrx 1和dmiRPrx 2中观察到严重的OA样表型。 dmiRAgc 1 ER KO小鼠和整个膝关节组织受到miR-204/-211缺失的影响，包括严重缺失 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- miR-204和miR-211是Runx 2表达的关键调节因子，在维持关节组织中发挥关键作用 体内平衡为了检验这一假设，提出了两个具体目标。在目标1中，我们将充分 在dmiRAgc 1 ER KO小鼠中表征miR-204/-211缺失对OA发展的纵向影响， 确定软骨细胞中Runx 2的缺失是否会逆转在dmiRAgc 1 ER KO小鼠中观察到的OA表型。在 目的2，我们将研究miR-204/-211在OA样本中表达的变化，并确定是否在体内 miR-204的施用将预防OA发展或减缓OA进展 小鼠模型这些拟议的研究将为OA发展机制提供新的见解， 将有助于开发新的OA治疗策略。

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.