miR-145通过TNFRSF11B/RANKL通路调节少关节型/全身型幼年特发性关节炎骨侵蚀的机制研究

Bone erosion is one of the most important factors affecting the prognosis of juvenile idiopathic arthritis (JIA)。 However, its mechanism remains unclear. MicroRNA-chip assay and our preliminary data showed that not only miR-145 was the plasma-specific increased microRNA in oligo-articular JIA (Oligo-JIA) and systemic JIA (sJIA)， but also miR-145 corrected with Juvenile Arthritis MRI Scoring for hips or knees, which were used to evaluated the degree of bone erosion via MRI imaging manifestations. The objectives of this project are 1) to further determine the expression level of miR-145 in the specimens of peripheral blood monocular cell, synovial fluid and synovium in Oligo-JIA and sJIA; 2) to explore the relationship between the differential expression of miR-145 and bone erosion in Oligo-JIA and sJIA; 3) to identify TNFRSF11B is one of target gene in the progress of bone erosion in Oligo-JIA and sJIA; 4) to elucidated molecular mechanism that miR-145 mediates the process of bone erosion by targeting TNFRSF11B on the levels of molecule, cell and animal in vivo; 5) to illuminate the mechanism that how the inflammatory cytokine regulates the expression of miR-145; 6) to verify miR-145 is involved in the process of bone erosion in Oligo-JIA and sJIA via targeting TNFRSF11B/RANKL pathway. In a word, the research of miRNA-145/TNFRSF11B/RANKL pathway might contribute a novel strategy to bone erosion treatment and new drug development in Oligo-JIA and sJIA.

骨侵蚀是影响幼年特发性关节炎(JIA)预后的重要因素，但具体机制不详。在前期借助miRNA列芯片筛选出miR-145在少关节型/全身型JIA血浆中特异性高表达的基础上以及与关节骨侵蚀程度影像学指标正相关的线索，本项目拟进一步分析miR-145在外周血单个核细胞、关节液及滑膜组织中的表达水平，探索miR-145异常表达与少关节型/全身型JIA骨侵蚀的关系，明确miR-145靶向作用于TNFRSF11B参与骨侵蚀通路，并从分子、细胞至动物水平深入研究miR-145靶向免疫分子在骨侵蚀发生过程中的分子机制，并阐明炎症因子对miR-145自身表达的调控机制；最后明确miR-145与TNFRSF11B／RANKL通路的作用关系和参与少关节型/全身型JIA骨侵蚀过程. miR-145/TNFRSF11B/RANKL信号通路的研究可能为少关节型/全身型JIA骨侵蚀的治疗、药物研发提供新策略。

骨侵蚀是影响幼年特发性关节炎(JIA)预后的重要因素，但具体机制不详。本研究取得主要研究结果如下：.1. oligo-JIA及sJIA患儿外周血、关节液及滑膜组织中miR-145表达明显升高，与TNFRSF11B表达负相关、骨侵蚀正相关（p<0.05）。.2.TNFRSF11B/RANKL比值降低在JIA骨侵蚀中发挥重要作用。我们研究发现：在oligo-JIA及sJIA患儿的外周血及关节液及CIA小鼠血清中：TNFRSF11B低表达，RANKL高表达（p<0.05）。.3.通过荧光素酶实验证实TNFRSF11B为miRNA-145的作用靶点。.4.通过细胞学研究证实miR-145通过调控TNFRSF11B／RANKL信号通路参与oligo-JIA及sJIA骨侵蚀。与对照组相比，miR-145 mimics转染配体（TNF-a 500ng／ml或IL-1β100ng／ml）刺激的RAW264.7细胞第24h，TNFRSF11B mRNA及蛋白表达均明显下降，而RANKL、TNF-α、IL-1β，IL-6及IL-17 mRNA及蛋白表达均明显升高（p<0.05）。miR-145 mimics转染RANKL重组蛋白（50ng/ml）诱导的Raw264.7细胞第3d、5d，破骨细胞正向标志性基因（Trap、Ctsk及MMP-9）高表达及负向标志基因（CTR）低表达（p<0.05），TRAP染色观察到破骨细胞分化及骨吸收功能增加；转录因子（c-Fos、c-Jun、NFATc1）的mRNA及蛋白表达增高及ERK、p38、IκBα因子的磷酸化水平升高；另外，采用流式细胞检测到破骨细胞凋亡受到抑制。因此，结合文献，我们研究表明，miR-145靶向抑制TNFRSF11B的表达，使其与RANKL竞争性结合减少，从而增强RANKL与RANK结合，激活下游p38 MAPK通路及NF-κB通路，促进破骨细胞分化、抑制破骨细胞凋亡，最终促进骨吸收及骨侵蚀。.5.动物实验表明CIA小鼠体内中miRNA-145过表达组具有较高骨侵蚀发生率。病理结果及Micro-CT扫描证实miRNA-145过表达组小鼠骨密质面积减少（骨破坏增加）、呈现大量白细胞浸润。miRNA-145过表达组小鼠软骨组织中TNFRSF11B及CTR低表达，而RANKL、Trap、Ctsk及MMP-9高表达（p<0.05）。