miR-29a调控Wnt/β-catenin信号通路参与强直性脊柱炎发病机制的研究

Ankylosing spondylitis (AS) is a systemic disease mainly involving sacroiliac joint and the spine, generally affecting young male adults.Ankylosis in advanced stage might lead to disability, and effective treatment is not availabe now. Wnt/β-catenin signal pathway plays a critical role in AS pathogenesis,and Dkk-1 is an important negative regulator of Wnt/β-catenin signal pathway. Dkk-1 is disfunctional in AS. Blockade of Dkk-1 induces sacroiliac joint infusion and protects syndesmophte in AS. MicroRNA (miR-29a) is a positive regulator of osteoblast differentiation and our previous study showed that miR-29a expression is significantly higher in AS patients. Bioinformatic prediction and in vitro experiment proof that Dkk-1 is the target of miR-29a. Our study designs to detect the expression of miR-29a, Dkk-1 and related gene expression in peripheral blood mononuclear cell(PBMC) and osteoblast of AS patients and controls to investigate their correlation; to verify miR-29a can target Dkk-1 and susequently facilitate Wnt/β-catenin signal and osteoblast differentiation in vitro cell experiment and in vivo animal experiment. These results will provide novel experimental evidence to elucidate the mechanism of miR-29a participation in AS new bone formation and provide new thoughts for AS treatment in clinical practice.

强直性脊柱炎（AS）是以骶髂关节及脊柱受累为主的疾病，好发于青壮年男性，晚期可致残，目前无有效治疗。Wnt/β-catenin信号通路在AS成骨中起关键作用，Dkk-1是其重要负调控因子，在AS中异常表达，阻断Dkk-1可促进患者骶髂关节融合，对AS患者的骨赘形成起保护作用。miR-29a是成骨分化正调控因子，是生物信息学和体外细胞实验证实的靶向阻断Dkk-1的重要microRNA，前期研究表明miR-29a在AS患者中表达显著升高。本课题检测AS患者和对照者外周血单个核细胞和成骨细胞中miR-29a、Dkk-1及相关基因的表达，探究其相互作用；应用体外细胞实验和体内动物试验证明miR-29a靶向Dkk-1调控Wnt/β-catenin信号通路促成骨分化，研究miR-29a靶向DKK-1的调控区域，为阐明miR-29a参与AS新骨形成发病机制提供新的实验依据，为临床AS诊治提供新治疗靶点。

目的：阐明miR-29a可通过下调靶基因Dkk-1的表达激活Wnt信号通路参与强直性脊柱炎新骨形成的发病机制。.方法：分别在临床血清mRNA和蛋白水平、体外细胞调控水平和体内动物模型水平检测miR-29a、Dkk-1及Wnt信号通路相关因子的表达。.结果：AS组中血清Dkk-1浓度低于对照组，差异有统计学意义（P <0.05），AS组中血清β-catenin浓度高于对照组，差异有统计学意义（P <0.05），AS组中血清GSK-3β、OC浓度与对照组差别无统计学意义（P ＞0.05）。血清中Dkk-1与β-catenin呈负相关，P <0.05；与GSK-3β、OC、CRP、ESR、miR-29a均无相关性，P＞0.05；miR-29a与CRP、ESR的表达均无相关性。生物信息学和体外细胞实验均证实在AS中，LRP6是miR-29a的靶基因。miR-29a-3p能够抑制LRP6基因表达。29a mimics 与29a inhibitor尾静脉注射后分别出现较NC的miR-29a的表达升高和降低。 29a mimics与29a inhibitor注射入小鼠后相应的Dkk-1减少、LRP6升高、GSK-3β减少、β-catenin、Collagen X、ALP、OC和Runx2均表达升高，证实Wnt信号及相关效应因子的激活。.结论：AS中存在miR-29a的升高和经典Wnt信号的活化，可能解释AS新骨形成的部分机制。细胞调控和动物实验证实，miR-29a可以促使Wnt信号及相关效应因子的激活。