miR-185-5p介导的circ-FAM169A调控椎间盘退行性改变的作用及相关机制研究

Pathogenesis of intervertebral disc degeneration (IDD) includes up-regulated expression of matrix metalloproteinase (MMP) and degradation of extracellular matrix. Circular RNA (circRNA) can abrogate inhibition effect of miRNA on its target gene through competitive endogenous RNA (ceRNA), thereby regulating gene expression. Studies by the research group had indicated that, in nucleus pulposus cells, miR-185-5p may bridge circ-FAM169A and MMP-2 through ceRNA mechanism, based on which, the group proposed that circ-FAM169A/miR-185-5p/MMP-2 signaling pathway regulated degradation of extracellular matrix-collagen to affect IDD progress. Our group intend to explore mutual regulation effect of miR-185-5p-mediated circ-FAM169A and MMP-2 using western blot analysis, luciferase assay and other laboratory technologies; and establish overexpression/knockdown of circ-FAM169A in nucleus pulposus cells and intervertebral discs of IDD model mice to observe expression quantity of MMP-2 and changes in degradability of collagen matrix, and then conduct an imaging assessment. The study aimed to explore the regulatory effect of circ-FAM169A on IDD so as to provide IDD gene therapy with new ideas and approaches.

椎间盘退行性改变（IDD）的发病机制包括基质金属蛋白酶（MMP）的表达上调和胞外基质的降解。环状RNA（circRNA）可通过竞争性内源RNA（ceRNA）机制解除miRNA对其靶基因的抑制来调控基因表达。课题组研究提示：在髓核细胞内miR-185-5p可能通过ceRNA机制桥接了circ-FAM169A及MMP-2。基于此课题组提出：circ-FAM169A/miR-185-5p/MMP-2信号通路通过调控胞外基质-胶原蛋白的降解，影响IDD进程。本课题拟用免疫印迹、荧光素酶实验等技术探索miR-185-5p介导的circ-FAM169A与MMP-2相互调控作用；在髓核细胞和IDD模型鼠椎间盘内过表达/敲低circ-FAM169A，观察MMP-2表达量及胶原蛋白基质降解度变化，并行影像学评估。本研究旨在探索circ-FAM169A对IDD的调控作用，为IDD基因治疗提供新的思路和方法。

椎间盘退变(IDD)是引起腰痛及相关的退行性肌肉骨骼疾病的原因之一，其发病机制包括基质金属蛋白酶（MMP）的表达上调和胞外基质的降解。环状RNA（circRNA）可通过竞争性内源RNA（ceRNA）机制解除miRNA对其靶基因的抑制来调控基因表达。本课题组通过生信发现miR-185-5p作为退行性髓核组织中的关键靶点，并通过qPCR、WB、流式细胞实验等技术，证实miR-185-5p可以靶向MMP-2调节髓核细胞功能。此外还通过生物信息学分析预测到miR-185-5p具有circ-TIMP2的结合位点，并通过细胞转染、免疫印迹、荧光素酶等实验技术证实circ-TIMP2通过调节miR-185-5p和MMP2在NP细胞中发挥功能。在髓核细胞和IDD模型鼠椎间盘内敲低circ-FAM169A，可以显著增加Ⅱ型胶原蛋白和聚集蛋白聚糖的表达水平和降低椎间盘分级，证明circ-FAM169A对IDD的调控作用，为IDD基因治疗提供新的思路和方法。