miR-34a通过SIRT1-Notch-Sox9通路调控骨性关节炎软骨细胞增殖和分化的信号机制

Osteoarthritis (OA) is one of the most cripple disease in the skeletal system. With the progrseeive ageing of the population , it is becoming a major public health problem. Current clinical therapies such as pharmaceutical interventions, bone marrow stimulation or microfracture focus on the short-term relief of OA symptoms, but do not result in regeneration of healthy cartilage tissue. When pharmaceutical intervention fails, clinicians regularly use invasive and permanent solutions such as jiont replacement. The molecular targeted therapy has a great potential for OA patients, however, its regulation mechanism is still unclear. In our recent study, we found that the expression level of miR-34a was significantly increased, while that of its predicted target gene SIRT1 and Sox9 were significantly reduced during the onset or progression of OA. With the review of recent advances on OA studies, we hypothesize that miR-34a may regulate Notch pathway through its target gene - SIRT1 modulating the chondrocyte proliferation, differentiation and chondrogenesis in OA. To study the regulation mechanism of OA in miR-34a/SIRT1-Notch- Sox9 signaling pathway, we have established OA in vivo mouse model and Chondrocyte apoptosis in vitro model (exposure to IL-1β), the recombinant lentivirus will be performed to transfect target gene SIRT1, the expression levels of miR-34a, SIRT1, Notch1, Jagged1 and Hes1 will be detected using quantitative RT-PCR at different Mankin stages. The objectives of our study are to determine the key target genes in the SIRT1-Notch- Sox9 signaling pathway and explore regulation mechanism of the chondrogenisis in the development of progressive cartilage degeneration and osteoarthritis. The outcomes in this study will provide the scientific basis for further exploring new molecular targeted therapy for osteoarthritis patients.

骨性关节炎 (OA)是目前骨骼系统中最常见的致残疾病，由于缺乏有效治疗手段，多数患者最终只能进行关节置换。分子靶向治疗有望成为攻克OA的关键其临床应用前景广阔，但当前人们对分子调控OA发生发展的机制知之甚少。我们的前期研究发现在OA进展中miR-34a的表达量明显升高，其靶基因SIRT1和Sox9的表达显著降低；结合最新研究报道，我们推测miR-34a可能通过SIRT1介导Notch-Sox9信号通路，参与了OA软骨细胞增殖分化及软骨形成过程的调控。本项目拟通过大鼠骨性关节炎模型及软骨细胞培养体外实验，利用重组慢病毒技术及定量RT-PCR等手段研究OA不同Mankin分期miR-34a、SIRT1和Notch通路信号分子的变化及相互作用，以期揭示OA发生后软骨再生的主要调控路径和信号调控机制，探索分子靶向治疗的最佳位点，为利用分子靶向定点治疗关节系统疑难疾病提供新的理论依据。

骨关节炎的主要病理改变是软骨退变，软骨由软骨细胞和细胞外基质构成，其中软骨细胞的增殖、死亡及其功能的正常与否和细胞外基质代谢的平衡状态，对骨关节炎的发生发展有着直接的影响。课题组分别从滑膜间充质干细胞（SMSC）和过表达miR-140-5p的滑膜间充质干细胞（SMSC-140）中分离提取鉴定SMSC-Exos和SMSC-140-Exos，体内验证其对软骨细胞增殖、迁移、细胞外基质分泌的影响。并通过慢病毒、寡核苷酸、化学药物对软骨细胞进行干扰，深入到对其作用机理——非经典Wnt通路激活YAP——的阐明和验证。体内通过番红固绿与免疫组化来观察和评价外泌体对骨关节炎发展的干预效果。结果发现SMSC-Exos高表达Wnt5a、Wnt5b，通过非经典Wnt通路激活YAP，造成软骨细胞增殖、迁移能力增强，但是软骨细胞的正常细胞外基质形成功能受到了影响。过表达的miR-140-5p通过作用于RalA，缓解了YAP激活对SOX9及其下游软骨基质基因的抑制作用。从而在体内外实验中获得了更出色的表现，促进增殖、迁移，同时对软骨细胞外基质分泌的影响较小。项目从动物模型、细胞模型和信号通路三个方面探索了外泌体装载转运的miRNA在骨关节炎软骨细胞增殖、迁移、细胞外基质形成中的作用和分子机制。通过研究源自滑膜间充质干细胞的外泌体对骨关节炎治疗作用、出现与预期不相符的不良副作用，及其潜在的分子机理的研究；针对不良副作用提出优化方案，利用基因修饰和外泌体运载获得了效果更好的SMSC-140-Exos，并对其分子调控机制进行了初步研究，为后续通过其他途径调控目标靶点来获得更有潜力的预防药物的研究提供了基础理论依据。