Runx2及其靶向性MicroRNAs在后纵韧带骨化中的效应及机制研究

Ossification of the posterior longitudinal ligament (OPLL) is a common spinal disease with significant morbidity and severe consequence.The treatment is challanging. There is no certain conservative therapy or effective method up to now due to its unclear etiology.Our previous studies of the two continous projects of NSFC have demonstrated that changes in Runx2 is closely related to the onset of OPLL, and the expression of Runx2 in posterior longitudinal ligament cell can be regulated by miR-29 and miR-34, which indicated that miRNA may play a key role in the development and progression of OPLL by regulating Runx2. In our current proposal, we plan to use primary posterior longitudinal ligament cell from OPLL patients and OPLL mouse model to further investigate how Runx2 and its targeting miRNAs are connected with the onset of OPLL. We will test the hypothesis that Runx2-targeting miRNAs mediate the process of posterior longitudinal ligament cell ossification in vitro by overexpression or knockdown of miR-29 or miR-34, luciferase activity assay of target gene reporter, and immunohistochemistry or qPCR detection. Then the possible signal pathways involved in this process will be further investigeted. Our research could provide new insight into the mechanism of the deverlopment and progression of OPLL through a miRNAs mediated manner, and may throw light on the early conservative therapy or early dignosis.

脊柱后纵韧带骨化症（OPLL）发病率高，危害大，是临床棘手的问题。其发病分子机制不明，目前尚无有效干预及非手术治疗措施。课题组在前两个自然基金项目资助的基础上，发现Runx2基因表达水平与OPLL的发生密切相关，并且发现mir-29，mir-34能够在后纵韧带细胞骨化过程中调控Runx2的表达，提示miRNAs介导的Runx2表达可能是促进OPLL进展的重要机制。本项目组拟围绕Runx2及其调控miRNAs，通过研究患者原代培养细胞及动物模型中Runx2及其调控miRNAs对OPLL发生发展的影响,明确Runx2及其靶向miRNAs与OPLL发生的相关性；通过基因过表达和干扰实验、报告基因实验、骨化相关因子的检测等验证其对后纵韧带细胞骨化的影响；应用表达谱分析,探讨miRNAs参与OPLL发生的关键信号通路，深入研究其机制作用，为明确OPLL的发生机理、早期诊断、早期干预打下基础。

本项目延续自申请人前两项国家自然科学基金项目（30600630、81171677），针对脊柱后纵韧带骨化症（OPLL）进行病因机制研究。针对前期发现中国人群中OPLL患者Runx2基因的SNP变化与该疾病发生的相关性，进一步探索找到其SNP可能对Runx2表达上游调控分子microRNA的作用产生影响，进而促进骨化的形成。通过本项目的实施，课题组在前期研究基础上针对OPLL及正常脊柱韧带组织之间microRNA区别的研究在国际上未见报道，首次进行了OPLL组织的microRNA高通量测序分析，共检测出1520条微小RNA，其中218条微小RNA具有明显的差异表达趋势。通过深入研究，发现microRNA-10a具有抑制ID2促进OPLL进展的作用；发现miR-563具有抑制SMURF1促进骨化的作用；发现miR-129具有抑制SMAD减少骨化进程的作用。此外在转化方面，利用该数据结合患者外周血清microRNA的表达检测，发现4种具有OPLL诊断价值的microRNA。课题组在本项目实施中共发表SCI论著3篇，在投2篇，发表核心期刊论著3篇，申请国家发明专利1项，国际PCT发明专利1项，为后续针对OPLL发生机制的进一步深入研究奠定重要的理论基础，也为今后OPLL的诊疗带来新的思路。