DLX3通过lncRNA-ENSG00000237125.3调控骨衰老的分子机制研究

Bone aging-related disease such as osteoporosis and severe absorption of alveolar ridge are the major clinical challenges. Tricho-dento-osseous (TDO) syndrome, characterized by increased bone mass and density, is an autosomal dominant hereditary disease caused by mutation of distal-less homeobox 3 (DLX3) gene, which is an ideal model for gene regulation of bone senescence. Previous studies in our research have showed that the bone marrow mesenchymal stem cells (TDO-BMSCs) extracted from patients with TDO syndrome (DLX3-Q178R) exhibits slower senescence rate. Cell transfection in vitro and transgenic animal in vivo experiments show that DLX3 knockdown or mutation delays bone senescence. However, the molecular mechanism by which DLX3 regulates bone senescence is unclear. In this view, this study screens out lncRNA-ENSG00000237125.3 by high-throughput sequencing analysis with the subject of TDO-BMSCs, suggesting that it may be one of the key targets for the regulation of bone senescence. Therefore, this study intends to explore the function and molecular mechanism of lncRNA-ENSG00000237125.3 in the regulation of BMSCs senescence, and explore the function and molecular mechanism of DLX3 in the regulation of senescence of BMSCs through the key factor lncRNA-ENSG00000237125.3. This study will provide new perspective and targets for the prevention and treatment of bone aging-related diseases.

骨衰老相关疾病如骨质疏松、牙槽嵴的重度吸收等是临床面临的重大挑战。毛-牙-骨（TDO）综合征是由DLX3基因突变导致的骨量及骨密度增加的显性遗传病，为骨衰老的基因调控提供了理想的研究模型。本课题前期研究发现TDO综合征患者（DLX3-Q178R）提取的骨髓间充质干细胞（TDO-BMSCs）衰老明显减缓，细胞转染和转基因动物实验显示DLX3敲低和突变可以延缓骨衰老。然而，DLX3调控骨衰老的分子机制尚不清楚。鉴于此，本课题对TDO-BMSCs行高通量分析，筛选出表达差异显著的lncRNA-ENSG00000237125.3，提示其可能是调控骨衰老的关键靶点之一。因此本研究拟通过探讨lncRNA-ENSG00000237125.3调控BMSCs衰老的功能和分子机制，探索以其为核心分子，DLX3调控BMSCs衰老的功能和分子机制。本研究将为实现骨衰老相关性疾病的防治提供新的思路和靶点。

由DLX3单基因突变导致的毛-牙-骨（TDO）综合征患者存在颅骨和颌骨骨密度渐进性增加的临床有利骨表征，为研究抵抗牙槽骨骨吸收和骨质疏松症等骨代谢相关疾病提供了良好的模型。本项目对比研究正常人和TDO患者来源的骨髓间充质干细胞的生物学行为差异及分析其非编码RNA表达谱高通量数据时发现，TDO综合征患者（DLX3-Q178R）提取的骨髓间充质干细胞（TDO-BMSCs）衰老明显减缓，且长链非编码lncRNA-ENSG00000237125.3和H19等基因表达在正常人和TDO患者来源的BMSCs表达存在明显差异，且lncRNA-ENSG00000237125.3与人长链非编码基因HAND2AS1同源。我们随后通过过表达及敲低lncRNA-ENSG00000237125.3、HAND2AS1和H19，以及ALP染色、茜素红染色、microRNA表达、RIP实验、荧光素酶报告基因、Real-time PCR和western blot等实验，发现了新机制（1）DLX3突变通过H19/miR-29c-3p/KDM5B轴调控成骨分化；（2）DLX3通过Myf5通过成肌分化，以及（3）HAND2OS1通过DKK1调控成骨和成肌分化的新分子机制。为了进一步挖掘TDO-BMSCs的价值，我们利用仙台病毒将SOX2、KLF4、OCT4和c-MYC 基因转染至TDO-BMSCs，构建了TDO-iPS细胞，通过体外3胚层分化及体内形成崎胎瘤实验，证实TDO-iPS构建成功，并且证实TDO-iPS衍生的BMSCs，具有与TDO-BMSCs相同的成骨分化特征，即弱化了的成骨分化能力。本项目所构建的TDO-iPS细胞可以解决TDO-BMSCs的有限来源问题，为将来利用TDO-BMSCs提供了物质基础。