miRNA-224调控Wnt/β-catenin信号通路在激素性股骨头坏死中的机制研究

Disorders of cell activity and differentiation of bone marrow mesenchymal stem cells (BMSCs) induced by glucocorticoids play an important role in steroid induced osteonecrosis of the femoral head. Wnt/β-catenin signaling pathway is confirmed as one of the important signaling pathways involved. Recent literature search and our previous work suggest that this pathway might be regulated by intrinsic miRNA-224. In order to prove this idea, this project aims to study under different concentrations of hormones, the change regularity of miRNA-224 in BMSCs and the influence on Wnt/β-catenin signaling pathway. Then with the construction of miRNA-224 over expression or inhibition lentivirus vector, as well as dual luciferase reporter analysis to confirm the target gene, we aim to clarify the regulatory mechanism of miR-224 in Wnt/β-catenin signal pathway, and its effect on BMSCs proliferation and osteogenic differentiation. Finally, through the establishment of rabbit model of osteonecrosis, this project will investigate the therapy effect of over expression or inhibition of miRNA-224 on steroid induced osteonecrosis of the femoral head. The aim of this study is to clarify the pathophysiological mechanism of miR-224 in the pathogenesis of steroid induced osteonecrosis, and to explore a new target for the treatment of steroid induced osteonecrosis.

糖皮质激素诱导的骨髓间充质干细胞（BMSCs）活性及分化方向的改变在激素性股骨头坏死中发挥重要作用，其中Wnt/β-catenin信号通路被证实为参与其中的信号通路之一。最新文献和我们的前期工作提示这一通路可能受到内在miRNA-224的调控。为证明该设想，本项目拟研究在不同浓度激素的作用下，BMSCs中miRNA-224的变化规律及对Wnt/β-catenin信号通路的作用；进一步通过构建miRNA-224过表达或抑制慢病毒载体，结合双荧光素酶报告分析验证其靶基因来阐明miRNA-224对Wnt/β-catenin通路调控的具体分子机制，以及对BMSCs增殖和成骨分化方向的影响；最后通过建立大白兔骨坏死模型，初步探讨体内miRNA-224过表达或抑制对激素性股骨头坏死的修复作用。本研究旨在明确miRNA-224在激素性骨坏死发生中的病理生理机制，探索一种治疗激素性骨坏死的新靶点。

糖皮质激素诱导的骨髓间充质干细胞（BMSCs）活性及分化方向的改变在激素性股骨头坏死中发挥重要作用，最新文献和我们的前期工作提示这一通路可能受到内在miRNA-224的调控。本项目拟明确miRNA-224在激素性骨坏死发生中的病理生理机制，探索一种治疗激素性骨坏死的新靶点。本项目取得以下研究成果：1）在激素作用下，BMSCs中的miR-224表达明显上调。在功能实验中，通过构建miR-224 mimics, miR-224 inhibitor发现miR-224可以抑制BMSCs的成骨作用，增强BMSCs的成脂作用。2）通过生物信息学及后续双荧光素酶报告分析确证了Smad4为miR-224的直接靶基因。此外通过分子生物学实验确证了Smad4-Taz轴为miR-224调控BMSCs分化的分子信号通路。进一步的，确证了Smad4-Taz-RUNX2为miR-224抑制BMSCs成骨分化的信号通路，Smad4-Taz-PPARγ为miR-224促进BMSCs成脂分化的信号通路。3）成功构建了激素性ONFH动物模型，改良了miR-224 antagomir的注射方式，结果发现miR-224 antagomir可以提高股骨头内新生骨形成，抑制脂肪形成，进一步分析确证miR-224 antagomir较对照组可以明显降低激素性ONFH的坏死率。本项目较为系统的阐明了miR-224在激素性ONFH发病机理中的相关作用，提出了miR-224对BMSCs的成骨-成脂双向调节作用，相关成果已被骨科经典SCI杂志《Bone》(JCR 1区)接收。