miRNA－21对牙龈上皮细胞内TLR2信号通路的调控机制研究

Human Periodontal disease is generally accepted to be a chronic infectious disease characterized by the interaction between gram-negative bacteria and host inflammatory responses, which results in the loss of alveolar bone and the destruction of connective tissues. TLR2 and its related signalling pathway play an essential role in initiating periodontal immune inflammatory responses by periodontal immune cells, as well as in the causation of periodontitis.The search for negatively regulatory mechanisms of TLR2 sigalling pathway will provide critical information on the periodontal pathogenesis.Strong evidence showed that the expression level of TLR2 was closely correlated with periodontal disease activity. TLR2 and its signalling pathway was modulated by various molecules, such as microRNA-21. It has been demonstrated that miRNA-21 could control innate immune responses by tageting the molecules of TLR2 signaling pathway. However, the precise roles of miRNA-21 and its reactions with TLR2 signalling pathway in periodontitis are poorly understood. The present project aims to explore the molecular mechanisms of miRNA-21 regulating TLR2 signaling pathway induced by P.g LPS in human gingival epithelium using in silico analysis, mRNA microarray and luciferase assay. Successful elucidation of the roles and reactions between miRNA-21 and TLR2 in peridodntal tissues, and how these molecules are modified by periodontal pathogens, will offer new insights into periodontal pathogenesis and facilitate the rational design of therapeutic interventions.

TLR2信号通路是牙周致病菌激活牙周细胞免疫反应的主要途径之一，但是过度的TLR2信号是导致牙周炎发生的重要因素，深入研究TLR2信号通路的负性调控机制是破解牙周炎病因的关键所在。申请者发现，TLR2信号通路受多种分子调控，其中miRNA－21以网络化方式对该通路进行负性调控，从而在炎症起始阶段控制整个免疫应答的范围和强度，防止过度炎症发生。但是miRNA-21在牙周非免疫细胞中调控TLR2信号通路的具体细节及机制尚不清楚。本项目旨在采用基因沉默和mRNA芯片等方法筛选牙龈上皮细胞内TLR2信号通路中受miRNA-21调控的靶基因,利用荧光酶素基因报告验证miRNA-21的作用位点与各靶基因3'UTR 的关系,分析miRNA-21对TLR2信号通路的调控方式，进而阐明miRNA-21调控TLR2 信号通路的分子机制，为利用miRNA靶向干预或治疗牙周炎，清除牙周致病菌提供更多科学依据。

TLR2信号通路是牙周致病菌激活牙周细胞免疫反应的主要途径之一，目前发现，TLR2信号通路受多种分子调控，其中miRNA－21以网络化方式对该通路进行负性调控，从而在炎症起始阶段控制整个免疫应答的范围和强度，防止过度炎症发生。Smad7蛋白是转化生长因子β（TGFβs）超家族重要的细胞因子，担负着调节细胞生长、分化、凋亡等过程，可通过可逆磷酸化对多种信号传导通路的功能起着关键的调节作用。 PDCD4作为一种抑癌基因，可直接与靶基因的mRNA编码区域结合阻断翻译过程，或通过与真核细胞翻译起始因子4G竞争性结合eIF4A，抑制靶蛋白的翻译起始，进而影响细胞的增殖、凋亡、转化、侵袭及自噬等生物学行为;在促进炎症的发生，在肿瘤、肥胖、糖尿病和心血管等疾病的发生发展中发挥重要作用，而且已有研究证实miR-21介导的PDCD4下调是AP-1活性最大化的必要条件。本项目采用基因沉默和mRNA芯片等方法筛选牙龈上皮细胞内TLR2信号通路中受miRNA-21调控的靶基因,利用荧光酶素基因报告验证miRNA-21对Smad7与PDCD4的调控方式，结果发现①与正常牙龈上皮细胞相比，miR-21 在P.g LPS组中的相对表达量明显上调（P<0.05），但miRNA-21的表达随P.g LPS 浓度的升高而升高，但与刺激时间无关，因此初步认为具有剂量依懒性。②miRNA-21 过表达慢病毒体外转染牙龈上皮细胞的转染率在10%以下，但在受P.g LPS激活的牙龈上皮细胞中的转染率高达50%。③无论有无P.g LPS刺激，牙龈上皮细胞转染过表达慢病毒后，miRNA-21 的表达均显著上调（p＜0.05），且P.g LPS 诱导组的表达显著高于未刺激组。④miRNA-21分别可与SAMD7，PDCD4发生作用,提示Smad7，PDCD4可能是 miRNA-21 作用的靶基因之一。⑤双荧光素酶报告基因验证miR-21 可以抑制SMAD7基因, PDCD4基因3′UTR 报告基因载体的荧光素酶活性（P < 0.05）。从以上结果发现，miRNA-21在牙周非免疫细胞中可能通过抑制SMAD7基因, PDCD4基因3′UTR达到负性调控TLR2信号通路。