miR-23a和miR-130a靶向调控Cx43及相关信号通路在颈椎后纵韧带骨化发生过程中的作用机制研究

Cervical ossification of the posterior longitudinal ligament (OPLL) is one of the most common spinal diseases and its pathogenic mechanism has not been fully understood. Our previous studies of the first project of NSFC have demonstrated that Cx43 promoted osteogenic differentiation of ligament cells partly through MAPK signaling pathway in the development of OPLL. However, it is still not clear about regulating mechanism of Cx43 expression, and other signaling pathways are also involved in the process of osteogenic differentiation of ligament cells. Our previous research has screened out miR-23a and miR-130a which may play a role in regulation of Cx43 expression and osteoblastic differentiation of ligament cells, based on the data of gene arrays and bioinformatic analysis. In this study, we will employ the cells from cervical posterior longitudinal ligament and the mechanical strain induced osteoblastic differentiation model, to investigate the role of miR-23a and miR-130a on regulation of Cx43 and related signaling pathways in the process of OPLL development. The results will also be tested in clincial tissue specimen and animal models. This research programme will elucidate the upstream and downstream regulation mechanism of Cx43 expression in the process of OPLL, and establish the Cx43-centered signaling pathways in osteogenic differentation of posterior longitudinal ligament cells. It will help to provide a reliable target for drug and gene therapy in OPLL.

颈椎后纵韧带骨化症（OPLL）是常见的脊柱疾患，但发病机制尚不完全清楚。课题组在前一个项目研究中发现，OPLL发生过程中Cx43通过MAPK信号通路调控后纵韧带细胞成骨分化。然而Cx43的上游调控机制并不清楚，Cx43介导的细胞成骨分化作用还可能涉及其它信号通路。前期研究已采用基因芯片和生物信息学分析筛选出miR-23a和miR-130a可能参与调控Cx43表达和细胞成骨。本课题拟以体外培养的颈椎后纵韧带细胞为研究对象，以机械应力诱导细胞成骨分化为模型，通过多种分子生物学方法对miR-23a和miR-130a调控Cx43和相关信号通路在OPLL过程中的作用机制进行深入研究，并通过临床组织样本和动物实验模型进行验证。该课题研究可进一步明确OPLL发生过程中Cx43的上下游表达调控机制，构建以Cx43为中心的后纵韧带组织细胞成骨分化信号调控网络，为OPLL的药物和基因治疗提供可靠靶点。

颈椎后纵韧带骨化症（OPLL）是常见的脊柱疾患，其发病机制尚不完全清楚。项目组前期研究发现缝隙连接蛋白Cx43在OPLL发生尤其是颈椎后纵韧带组织细胞成骨分化过程中发挥重要调控作用，但其Cx43基因和蛋白表达上游调控机制尚不明确。近年来，非编码RNA对其靶向基因的表达调控研究日趋成熟，但其在OPLL发生过程中的作用和调控机制研究甚少。. 由于正式试验与预实验结果有较大偏差，经过对课题重新设计，并结合前期lncRNA基因芯片检测和miRNA高通量测序结果，项目从细胞和组织学层面研究了lncRNA-miRNA相互作用对Cx43基因和蛋白表达调控及相关信号通路在颈椎后纵韧带骨化发病机制中的作用机制。.（1）通过高通量测序及基因芯片技术，检测OPLL和非OPLL患者miRNA和lncRNA的表达差异，通过文献综述和生物信息学分析，筛选可能对Cx43基因或蛋白表达调控的miRNA和lncRNA分子；.（2）通过Western-blot、qPCR、荧光免疫报告实验等分子生物学实验验证了lncRNA MALAT1可以通过“海绵吸附”miR-1，靶向调控Cx43基因表达和下游NFkB信号通路，从而对颈椎后纵韧带细胞成骨分化进行调控；.（3）同样通过多种分子生物学实验验证lncRNA XIST可以通过“海绵吸附”miR-1miR-302a-3p，靶向调控Cx43的去泛素化蛋白酶USP8的基因表达，从而实现对Cx43蛋白表达水平的调控；此外，研究还发现miR-302a-3p靶向作用m6A甲基转移酶METTL3反向调控lncRNA XIST表达，实现lncRNA-miRNA相互作用。. 项目研究阐明lncRNA-miRNA相互作用在OPLL发病机制中的作用，并构建颈椎后纵韧带组织细胞成骨分化过程中以Cx43为中心的调控网络, 并从m6A甲基化修饰表观遗传学角度进一步拓展了OPLL发病机制的研究。