LncRNA MEG3与BMP-9诱导间充质干细胞成骨分化的关系研究

Bone morphogenetic protein 9 (BMP-9) is the most powerful growth factor in the BMPs members which induce the osteogenic differentiation of mesenchymal stem cells (MSCs). It possesses a wide prospect of clinical application. However, as its mechanism is complex and does not follow the traditional classical BMP signal transduction pathway, it is difficult to analyze the osteogenic differentiation mechanism of BMP-9 roundly by using the existing research methods. We have firstly structured cells which resist the osteogenic induction of BMP-9 by using a genome-wide small molecule RNA library original made by us. The resistance of cells has been confirmed by the studies in vitro. Compared with control group, we found that the expression of long noncoding RNA MEG3 (lncRNA MEG3) was significantly down-regulated through RNA-HiSeq analysis. The cells can be induced to osteogenic differentiation partly by overexpression of lncRNA MEG3. Based on previous work, we will apply various experimental techniques to analyze the relationship between lncRNA MEG3 and the osteogenic differentiation induced by BMP-9 in MSCs through a series of studies in vitro and in vivo; and the conceivable molecular mechanism of lncRNA MEG3 involved in regulating osteogenic differentiation induced by BMP-9. The results of this study will contribute to enhance osteogenic differentiation mechanism of MSCs induced by BMP-9, and promote the development of bone tissue engineering mediated by BMP-9.

骨形态发生蛋白9(BMP-9)是已知BMPs成员中诱导间充质干细胞(MSCs)成骨能力最强的生长因子，具有广阔的临床应用前景。但其作用机制复杂且又不完全遵循传统经典BMP信号传导途径，现有研究方法难以全面分析。课题组前期应用自建的全基因组小RNA文库，首次获得对BMP-9成骨分化诱导耐受的干细胞，并经体外实验证实；经RNA-HiSeq分析，我们发现在该细胞中LncRNA MEG3的表达水平明显低于对照组，而过表达LncRNA MEG3后可以部分恢复该细胞的成骨分化能力。本项目在前期工作基础上，拟应用多种实验技术，通过一系列体外和体内实验，分析lncRNA MEG3与BMP-9诱导MSCs成骨分化的关系，以及lncRNA MEG3参与调节BMP-9成骨分化诱导功能的可能分子机制。本研究结果将有助于深入诠释BMP-9诱导MSCs成骨分化的机制，推动BMP-9介导的骨组织工程发展。

骨形态发生蛋白9(BMP-9)是已知BMPs成员中诱导间充质干细胞(MSCs)成骨能力最强的生长因子，具有广阔的临床应用前景。但其作用机制复杂且又不完全遵循传统经典BMP信号传导途径，现有研究方法难以全面分析。课题组前期应用自建的全基因组小RNA文库，首次获得对BMP-9成骨分化诱导耐受的干细胞，并经体外实验证实；经RNA-HiSeq分析，我们发现在该细胞中LncRNA MEG3的表达水平明显低于对照组，而过表达LncRNA MEG3后可以部分恢复该细胞的成骨分化能力。此次，我们以BMP9诱导的骨髓间充质干细胞作为成骨模型，以探讨MEG3对成骨的影响；体内实验采用异位成骨和组织学染色；碱性磷酸酶（ALP）染色和茜素红染色用于成骨评价；竞争性内源性RNA（ceRNA）网络分析和双荧光素酶报告实验用于具体的机制研究。实验结果表明，过表达MEG3可以促进BMP9诱导成骨标志物、ALP活性和基质矿化。敲除MEG3可减弱BMP9诱导的成骨标志物表达。MEG3促进了GSK-3β的磷酸化和β-catenin的蛋白表达水平。与此同时，丙酮酸脱氢酶激酶4（PDK4）也能与GSK-3β结合，并促进后者的磷酸化，而MEG3又可以提高PDK4的mRNA表达水平。ceRNA网络分析表明，microRNA 532-5p（miR-532-5p）参与了MEG3对PDK4的作用。miR-532-5p可减弱MEG3增强的GSK-3β/β-catenin信号轴，miR-532-5p抑制剂可部分挽救内源性PDK4和MEG3介导的PDK4表达。MEG3和PDK4可协同增强成骨GSK-3β/β-catenin信号轴。综合以上数据，我们发现MEG3可能通过抑制miR-532-5p而增强PDK4和GSK-3β/β-连环蛋白。