MicroRNA-145调节骨关节炎软骨胞外基质代谢失衡的作用及机制研究

Osteoarthritis (OA) is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multi-factorial, the cartilage destruction appears to be a result of the failure of the chondrocytes to maintain a balance between extracellular matrix (ECM) anabolism and catabolism. However, the molecular mechanisms involved in OA remain unclear. In previous study, we found that microRNA-145 was significantly up-regulated in OA cartilage by quantitative PCR. To investigate the function of miR-145 in OA cartilage, we performed the loss of miR-145 function study to examine whether the expression of the OA-related genes regulated by miR-145, when chondrocytes were stimulated with IL-1β. The results showed that the expression of MMP-13 and ADAMTS-5 with IL-1β stimulation was significantly reduced by anti-miR-145. Conversely, the expression of Aggrecan , Col2a1 and COMP was significantly increased. The results suggest that miR-145 plays an important role in regulating the balance between anabolism and catabolism of ECM. Sox9 and Smad3, as an essential co-activator of Sox9, are both important factors in OA pathogenesis. Based on our previous study that miR-145 directly regulates Sox9 expression on protein level and Smad3 is also a potential target of miR-145, we propose a hypothesis that miR-145 may control articular cartilage homeostasis in OA through directly regulating Sox9 expression, meanwhile mediating the DNA-binding activity of Sox9 via targeting Smad3. In this project, we will perform the dual-luciferase report assay and in situ hybridization (ISH) to identify that miR-145 directly targets Smad3. Subsequently, under gain-of-function and loss-of-function assay, we will investigate how miR-145 regulates the balance of the ECM formation and degradation of OA cartilage in vitro and in mouse model of OA. Additionally, we will identify that miR-145 can mediate the DNA-binding activity of Sox9 via regulating Smad3 expression by co-immunoprecipitation. This research will provide new insights into the disease mechanisms and therapeutic interventions for OA.

关节软骨细胞外基质（ECM）代谢失衡是导致骨关节炎（OA）软骨退变的重要原因之一。我们发现OA关节软骨细胞中miR-145表达上调，抑制其能促进OA样软骨细胞表达特征型ECM基因，提示其参与调控OA关节软骨ECM代谢失衡，但机制不明。在证实miR-145调控Sox9蛋白表达的基础上，基于Sox9的辅激活因子Smad3是miR-145预测靶基因，且Smad3与OA病程进展相关，推测miR-145通过调节Sox9蛋白表达，同时经Smad3调节Sox9与靶基因的结合，参与调控OA关节软骨ECM代谢。拟通过双荧光素酶、原位杂交明确Smad3是miR-145靶基因；在体小鼠OA模型和离体细胞中干预miR-145表达，观察其对Sox9、Smad3、关节软骨ECM代谢和OA病程的影响，免疫共沉淀明确其参与Smad3介导Sox9与靶基因的结合的机制，阐明上述假说。本研究将为OA病因学和临床治疗提供新线索。

骨关节炎(Osteoarthritis, OA)是一种以关节软骨变性、损伤为主要病理特征，累及全关节的慢性退行性疾病。目前研究认为，关节软骨细胞外基质合成与分解代谢失衡是导致OA关节软骨退变的重要原因之一。然而，调节 OA关节软骨胞外基质代谢稳态的分子机制尚未阐明。临床上对于OA晚期病变的治疗缺乏有效的药物治疗方案。因此，探讨调节 OA关节软骨胞外基质代谢失衡的分子机制对于如何有效的预防、缓解OA病程进展具有重要的研究意义。基于我们之前的研究结果，我们推测microRNA-145可能是影响OA关节软骨胞外基质代谢失衡的重要因子，是导致OA关节软骨退变的重要miRNA，但miR-145如何调节软骨胞外基质代谢的具体分子机制不明。为此，本项目通过探讨miR-145调节OA关节软骨胞外基质合成与分解代谢失衡的作用及机制，进一步明确miR-145在OA病理进程中的生物学功能。.本项目主要研究内容及重要发现是：1、在正常关节软骨细胞和OA关节软骨细胞中，miR-145表达随interleukin-1 beta（IL-1β）刺激而显著升高。提示IL-1β是OA病程中miR-145表达异常的刺激因子。2，通过双荧光素酶等实验证实Sox9辅激活因子 mothers against decapentaplegic homolog 3（Smad3）是miR-145的靶基因。3、GOF和LOF功能实验证实，miR-145能通过靶基因Smad3对OA软骨细胞外基质代谢起调节作用。4、建立OA小鼠模型，体内实验显示miR-145是软骨细胞外基质代谢负向调控因子。.本项目研究结果阐明了miR-145通过直接调控Sox9蛋白表达，和以靶基因Smad3介导的间接途径调节OA关节软骨胞外基质代谢的分子机制；明确了miR-145通过调节OA关节软骨胞外基质代谢影响OA病理进程的重要作用。研究结果将有助于更深入理解OA关节软骨胞外基质代谢失衡的分子机制，为临床OA治疗靶点的研究提供新思路。