miR-335-5p靶向SP1调控髁突软骨成骨分化的作用及机制研究

Temporomandibular joint(TMJ) anterior disc displacement(ADD) is one of the most common TMJ diseases, which prefers to adolescent. ADD can increase the pressure on the surface of condyle and inhibit entochondrostosis of condyle, resulting in dentofacial deformity. However the mechanism of higher abnormal pressure leading to entochondrostosis dysfunction is not clear. The current studies have found that ADD(abnormal higher pressure)can up-regulate miR-335-5p and down-regulate ALP and osteocalcin. By data mining of three important databases, we have found that SP1 is one the target of miR-335-5p. As we know, SP1 is the critical collaborative molecule of Osterix(Osx), which plays a key role in the progress of condyle entochondrostosis. Our studies have also revealed that higher abnormal pressure can up-regulate SP1 in protein level while have no influence in RNA level in the chondrocytes. Thus we propose that the increased expression of miR-335-5p in adolescent TMJ ADD chondrocytes can inhibit SP1 in the protein level after transcription, which reduces the complex of SP1 and Osx. As a result it can repress the progress of condyle entochondrostosis. By using the established rabbit TMJ ADD animal model and inducible SP1 conditional knockout mice in vivo, and using miR-335-5p down-regulating or SP1 overexpression in vitro, we will study the role of miR-335-5p /SP1 axis in regulating Sox9, Runx2, Osx and their corresponding signaling pathways. Collectively, this study aims to identify the mechanism of adolescent TMJ ADD entochondrostosis dysfunction.

颞下颌关节盘前移位是临床常见病，青少年好发。盘前移位可引起髁突表面压力增加，软骨内成骨障碍。目前关于髁突负荷增加影响软骨内成骨的机制尚不明确。课题组前期研究发现异常压力增加可上调软骨细胞内miR-335-5p，下调ALP和骨钙素等成骨相关因子。数据挖掘显示SP1是miR-335-5p可能作用靶点。文献报道SP1是成骨分化关键分子Osx发挥转录调控作用的协同分子。前期研究发现异常高压力可降低软骨细胞内SP1蛋白水平而不影响RNA表达。为此我们提出：青少年颞下颌关节盘前移位髁突软骨内miR-335-5p升高，转录后抑制SP1蛋白表达，SP1和Osx复合体减少，抑制髁突软骨成骨分化。为验证该假说，本项目拟综合运用各种分子生物学技术，系统分析miR-335-5p在髁突软骨成骨分化中的作用，重点探索SP1和Osx及其下游信号通路调控髁突软骨内成骨的机制，为揭开盘前移位导致髁突发育障碍之谜提供依据。

青少年颞下颌关节盘移位是一类发病率高、潜在危害大的常见病。关节盘前移位是引起髁突发育障碍，导致牙颌面畸形的重要因素之一。在本课题的资助下：（1） 体外实验结果表明miR-335-5p能够抑制软骨内成骨，首次利用miR-335抑制剂治疗大鼠髁突的骨关节炎，并取得理想疗效，该部分研究目前正在投稿阶段。（2）课题组从hPSCs通过神经棘诱导外胚层软骨细胞（ECCs）的双阶段诱导。在维持稳定的自我更新后，细胞停止增殖并分化为同质的软骨细胞群。移植后，该细胞系能够在体内形成类似于下颌髁突软骨的软骨样结构，可能有助于开发基于细胞的颅骨软骨损伤治疗（npj regenerative medicine，2022）。（3）通过进行miR-335-5p的转录组测序，发现miR-335-5p作用与于靶基因SP1，激活TGF-β/SMAD2/SMAD3信号通路，抑制髁突的软骨内成骨（投稿中）。通过本课题的研究，我们明确了miR-335-5p通过抑制靶基因SP1的表达，抑制髁突的软骨内成骨，通过在大鼠UAC模型中，治疗性抑制miR-335-5p，能够有效治疗软骨退行性变，为将来的临床转化奠定了坚实的基础。