LncRNA MEG3通过FOXO1调控下游炎症靶基因影响胸椎黄韧带骨化发生的机制研究

In recent years, The incidence of thoracic ossification of the ligamentum flavum (OLF) gradually increased. Inflammatory response is regarded as a significant pathophysiological change in OLF process. And FOXO1 plays an important role in this process. The recent studies show that FOXO1 affects inflammatory response via various regulatory pathway. However, reports regarding the effect of lncRNA to OLF remains rare. Our research displays that: TNF-α level in the serum of OLF patient witnessed a remarkable increase; in the aspect of tissue, comparing with ligament, the TNF-α expression of ossified ligamentum decreased, while the expression of lncRNA MEG3 increased. Meanwhile, TNF-α expression of ossification tissue was upregulated and the expression of lncRNA MEG3 experienced a obvious decline. Therefore, we propose the hypothesis that lncRNA MEG3 is able to regulate inflammatory signaling pathway through FOXO1 and thus regulating the ligamentum ossification process. To testify the hypothesis, we will use primary cell and tissue model to define the role of lncRNA MEG3 on inflammation in OLF process through the level of molecular, cell and tissue. The regulatory mechanism of lncRNA MEG3 on FOXO1 will be clarified. And the role of lncRNA MEG3 on ossification process will also be explored. The research of this project is supposed to lay foundation for exploring OLF inflammation mechanism and produce new insight on OLF prevention.

近年来，胸椎黄韧带骨化（OLF）发病率逐渐上升。炎症反应是OLF过程中重要的病理生理变化。FOXO1在此过程中起重要作用。研究显示FOXO1通过多种通路调节炎症反应。但lncRNA在OLF中的作用鲜有报道。我们研究发现：OLF病人血清中炎症因子TNF-α水平明显升高；与韧带相比，骨化韧带中TNF-α的mRNA表达降低，lncRNA MEG3的表达上升，骨化块中TNF-α表达升高，lncRNA MEG3表达下降。因此我们提出假说：lncRNA MEG3可能通过FOXO1调控炎症通路，进而调节韧带骨化过程。为验证这一假说，我们应用原代细胞和组织模型，从分子、细胞及组织水平明确lncRNA MEG3在炎症参与的OLF骨化中的作用，阐明lncRNA MEG3对FOXO1的调控机理，探讨lncRNA MEG3对骨化过程的作用机制。本研究将为揭示OLF的炎症发生机制奠定基础，为OLF防治提供新思路。

胸椎黄韧带骨化（Thoracic ossification of the ligamentum flavum, TOLF）是一种常见的脊柱韧带异位骨化症，可引起严重的胸椎管狭窄和脊髓病。该项目原计划应用原代细胞和组织模型，明确LncRNA MEG3调节炎症进而参与TOLF骨化进程的作用及其机制。项目获得后，我们首先应用RNA测序技术分析了不同骨化模式下胸黄韧带细胞的基因表达谱。GO分析表明，血管生成素-2（Angiopoietin-2, ANGPT2）在原发性胸黄韧带细胞向成骨分化过程中显著升高，因此我们将研究计划略作调整，进一步研究发现上调ANGPT2可增加未成熟骨化的TOLF细胞中Notch2和成骨标志物如Runx2、ALP及OCN的表达，敲减ANGPT2后TOLF细胞内Notch2通路和成骨过程则被抑制，这些发现证明ANGPT2通过Notch信号通路调节胸椎黄韧带细胞成骨分化（2018 PLoS One）。为进一步探讨TOLF疾病进展变化的机制，我们分别检测健康人和TOLF患者两种进展阶段（单节段与多节段）黄韧带组织的基因组DNA甲基化谱改变情况。结果显示单节段与多节段黄韧带组织的基因组中有65个甲基化差异位点，焦磷酸测序及q-PCR验证发现6个甲基化差异基因（SLC7A11, HOXA10, HOXA11AS, TNIK, HOTAIR, IFITM1）与黄韧带骨化的进展密切相关，其中HOXA11AS与HOTAIR均为LncRNA，这是我们首次发现LncRNA的甲基化可能在TOLF疾病进展中起重要作用，也为TOLF疾病发病机制研究提供新的思路（2020 J Cell Mol Med）。