lncRNA LINC00473靶向miRNA-23a调控骨髓间充质干细胞成骨成脂分化机制研究

Steroid-induced femoral head necrosis (SFHN) is a high rate disease, and the exact pathogenesis of SFHN is not clear and lack of effective and early treatment. Therefore, pathogenesis research of SFHN is of great significance. The regulation of lncRNA, miRNA and related signaling pathway play an important role in the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSC). In this study, we identified lncRNA LINC00473 related to osteogenic and adipogenic differentiation of BMSC on the basis of previous microarray screening. Preliminary experiment showed that LINC00473 could promote osteogenic differentiation and inhibit adipogenic differentiation of BMSC. Pre-Experiment found that LINC00473 can target and inhibit the expression of miR-23a and increase the expression of LRP5. Accordingly, we propose the hypothesis: LINC00473 regulates osteogenic and adipogenic differentiation of BMSC via Wnt /β-catenin signaling pathway by targeting miR-23a. To test this hypothesis, we will use human BMSC and a modal in rats with steroid-induced femoral head necrosis, adopt real time PCR, western blot, adenovirus vector transfection and siRNA to detect the mechanism of the effect of LINC00473 on the osteogenic and adipogenic differentiation of BMSC by targeting miR-23a from the molecular, cellular, tissues, and animal levels. This study will provide a new perspective for the further study on the early treatment of SFHN.

激素性股骨头坏死发病率高，发病机制尚不明确，缺乏早期干预措施，因此开展其发病机制研究意义重大。lncRNA、miRNA及相关信号通路在骨髓间充质干细胞（BMSC）成骨成脂分化中发挥重要作用。我们芯片筛选出BMSC成骨成脂分化相关lncRNA LINC00473，预实验显示LINC00473可以促进成骨抑制成脂，可以靶向结合并抑制miR-23a表达，同时LRP5表达增加，所以我们提出假设：LINC00473靶向miR-23a通过Wnt/β-catenin通路调节BMSC成骨成脂分化。为验证此假说，本课题拟通过人BMSC、大鼠激素性股骨头坏死模型，采用real time PCR、western blot、腺病毒载体转染、RNA干扰等多方法，从分子、细胞、组织及动物水平等多层次，探讨LINC00473靶向miR-23a调控BMSC成骨成脂分化的作用机制，为进一步研究股骨头坏死早期治疗提供新视角。

激素性股骨头坏死（SONFH）发病率高，发病机制尚不明确，缺乏早期干预措施，因此开展其发病机制研究意义重大。lncRNA、miRNA及相关信号通路在骨髓间充质干细胞（BMSCs）成骨成脂分化中发挥重要作用。本课题通过在临床组织标本、骨髓间充质干细胞、动物实验等多个层面，采用real time PCR、western blot、腺病毒载体转染、RNA干扰、免疫沉淀、MicroCT、MRI等多方法，系统研究了LINC00473靶向miRNA-23a-3p通过不同的信号通路调节骨髓间充质干细胞成骨、成脂分化及凋亡的作用机制。本研究分子实验发现，LINC00473可以激活miR-23a-3p/LRP5/Wnt/β-catenin信号通路促进BMSCs的成骨和抑制脂肪生成，同时通过激活miR-23a-3p/ PEBP1/ Akt/ Bad/ Bcl-2信号通路从地塞米松诱导的细胞凋亡中拯救BMSCs；LINC00473具有与miR-23a-3p靶向结合的位点，并能够通过AGO2蛋白介导的方式进行靶向结合，进而影响miR-23a-3p的表达和相关生物学功能。进一步的动物实验发现过表达LINC00473能够促进BMSCs的体外迁移，且温敏型PLGA水凝胶能够作为支架能够增加BMSCs的粘附，维持移植细胞的存活，有利于BMSCs向股骨头坏死区域的迁移；LINC00473修饰后的BMSCs联合温敏型PLGA水凝胶移植能够有效改善SONFH大鼠股骨头的水肿状态，促进软骨下骨坏死区域的修复，此方案为临床上治疗SONFH提供了新的策略。本研究立足于骨髓间充质干细胞，从lncRNA与miRNA的新视角揭示激素性股骨头坏死的发病机制，为股骨头坏死发病机制研究提供新的理论依据，为早期治疗的研究提供新思路。