强直性脊柱炎差异表达MicroRNA调控致病易感基因ANO6的骨代谢异常机制研究

Ankylosing Spondylitis (AS) is a disease resulted from inflammatory and abnormal bone metabolism and so far without specific diagnostic and therapeutic method. We have finished the following works previously: 1.Expression disorders of up-regulated miR-17-5p are most significant peripheral blood monocytes (PBMC) in AS patients. 2.Susceptibility genes of the disease ANO6 are functional targets of the microRNAs mentioned above respectively. We put forward a hypothesis: increased miR-17-5p might participate to the beginning and development of abnormal bone metabolism through targeting the two genes respectively in AS. Our study is designed as followed: 1. Patients’ bone marrow mesenchymal cells (BMSC) are stimulated in vivo respectively by the two miRNAs mentioned above, the activation of ossification and suppression effect to osteoclasts mediated by ANO6 will be observed. miR-17-5p over-expressed animal model will be built to observe the effect of how miR-17-5p regulates ANO6 mediated chondrosteosis and inflammation in BMP/IHH pathway and activated ectopic bone-formation in Wnt pathway. The expected performances of the study might discover a new mechanism of abnormal bone metabolism and inflammation in AS, which will supply theoretical evidence in explicating new targets for the intervention of disease development in AS.

强直性脊柱炎（Ankylosing Spondylitis，AS）因炎症与骨代谢异常使病人骨关节强直致残，至今无特异诊治方法。我们研究发现：1）AS患者PBMC的miR-17-5p显著升高；2）ANO6是AS新的易感基因，并且是miR-17-5p靶向功能基因之一。我们的科学假说是：表达升高的miR-17-5p靶向ANO6参与AS骨代谢异常发生和发展。我们拟通过：1）用上述miRNA刺激AS病人骨髓间充质干细胞（BMSC），观察介导ANO6在激活成骨和抑制破骨中的作用。2）构建miR-17-5p过表达动物模型，观察miR-17-5p调控BMP/IHH通路中ANO6等基因在软骨骨化和炎症以及Wnt通道激活成骨在异位骨化中作用；为阐明AS炎症和骨代谢异常新机制和提供干预AS骨结构破坏发展新靶点提供证据。

通过近几年研究开展，“强直性脊柱炎差异表达MicroRNA调控致病易感基因ANO6的骨代谢异常机制研究”项目完成部分预期计划：.背景：强直性脊柱炎是一种主要累及中轴关节的慢性炎症性疾病，炎症和骨代谢异常是其突出表现。多种因素参与其致病过程。我们前期研究发现强直性脊柱炎患者PBMC的miR-17-5p显著升高。因此本研究拟通过体内外实验进一步验证其可能参与强直性脊柱炎发病机制。. 主要研究内容和结果是：.1、相较健康对照者，强直性脊柱炎患者、放射学阴性中轴型脊柱关节炎患者外周血单核细胞miR-17-5p明显高表达。非甾体抗炎药和肿瘤坏死因子拮抗剂联合治疗明显降低nr-axSpA患者miR-17-5p的表达。.2、在细胞实验水平，通过转染技术过表达及抑制表达miR-17-5p，探索miR-17-5p对小鼠成骨细胞MC3T3-E1成骨分化及矿化影响。结论：miR-17-5p使成骨细胞成骨分化能力减弱。.3、MiR-17-5p靶基因预测及验证：利用生物信息学靶基因预测软件预测miR-17-5p的靶基因，并通过双荧光素酶报告基因验证。结论：miR-17-5p在转录后水平调控ANO6蛋白表达，双荧光素酶报告基因检测证实ANO6非miR-17-5p的直接靶基因。.4、构建miR-17-5p转基因过表达模型，检测骨代谢标志物表达及骨微结构。结论：转基因小鼠及野生型小鼠在血及骨组织alp、runx2表达无差异，microCT检测两组小鼠股骨远端骨微结构参数无明显变化。