lncRNA TSC22D1-AS1/miR-765/BMP6轴向调控成骨分化机制研究

From the perspective of lncRNA, the project follows the idea of the ceRNA regulation mode and aims to systematically describe osteogenic differentiation of lncRNA TSC22D1-AS1/miR-765/BMP6 axial regulation. This study is divided into two parts (in vitro and vivo). It will be as a study object that human bone marrow mesenchymal stem cells differentiate into osteoblast in vitro. We will identify the relationship between each other by using luciferase, fluorescence in situ hybridization, and qPCR. Furtherly, overexpression (interference) of target genes and target gene replenishment experiments are carried to analyze the osteogenic phenotype, gene expression of osteogenic characteristics and Smads protein phosphorylation level in order to evaluate osteogenic ability. In vivo, hMSCs with different expression levels of lncRNA TSC22D1-AS1, miR-765 and BMP6 are implanted into nude mice subcutaneously for ectopic osteogenesis. Osteogenic potency is evaluated using X-ray (molybdenum target), HE staining, hard tissue slice and the same molecular assay with vitro. This study will systematically elaborate that lncRNA TSC22D1-AS1/miR-765/BMP6 (axial action) regulated the osteogenic differentiation mechanism via the BMP6/Smads signaling pathway in vitro and vivo. It will provide a new ideas and methods for profound understanding and treatment of senile osteoporosis.

项目以lncRNA为视角，沿着ceRNA调控模式的思路，朝着系统阐述lncRNA TSC22D1-AS1/miR-765/BMP6轴向调控成骨分化过程的目标开展工作。研究由体内外两部分组成，体外以成人骨髓间充质干细胞成骨分化为对象，运用荧光素酶、荧光原位杂交和qPCR等技术鉴定相互间靶向关系，采取目标基因过表达（干扰）、靶基因回补等方法，分析成骨表型特征、成骨特异性基因表达和Smads蛋白磷酸化水平评价成骨能力；体内研究将不同表达水平lncRNA TSC22D1-AS1、miR-765和BMP6的hMSCs植入裸鼠皮下进行异位成骨，运用X线（钼靶）、HE染色、硬组织切片和体外相同的分子检测评估成骨效能。从体内外系统阐述lncRNA TSC22D1-AS1/miR-765/BMP6以轴向作用方式，经BMP6/Smads信号通路调控成骨分化的机制，为认识和治疗老年性骨质疏松提供新的思路和方法。

随着人口老龄化加剧，骨质疏松症已成为社会面临的重要公共健康问题，人骨髓间充质干细胞（H-BMSCs）成骨分化能力减弱是其发病的主要原因之一。项目以lncRNA为视角，沿着ceRNA调控模式的思路，朝着系统阐述lncRNA TSC22D1-AS1/miR-765/BMP6轴向调控成骨分化过程的目标开展工作。具体内容如下：1）发现lncRNA TSC22D1-AS1和lncRNA FOXD2-AS1在成骨分化过程中表达显著增加，经筛选确定了过表达或敲低lncRNA FOXD2-AS1能显著影响成骨分化。进一步，过表达lncRNA FOXD2-AS1可显著提高了JAK2/STAT3的磷酸化水平，而敲低该lncRNA则能抑制JAK2/STAT3信号通路的磷酸化水平；同时，lncRNA FOXD2-AS1过表达后加入JAK2/STAT3抑制剂AG490进行回补实验，发现与lncRNA FOXD2-AS1过表达组相比，成骨分化受到抑制，且JAK2/STAT3磷酸化水平降低，以上结果表明，lncRNA FOXD2-AS1通过激活JAK2/STAT3信号通路驱动成骨分化。2）利用敲低或过表达miR-765均可明显影响成骨分化；通过双荧光素酶实验验证了miR-765与靶基因BMP6的3'-UTR具有结合位点，证实了miR-765靶向作用BMP6调控成骨分化。同时，分析miR-765表达改变对Smad1/5/9磷酸化水平影响，以及在miR-765敲低组添加 Smad1/5/9信号抑制剂，发现与miR-765敲低组相比成骨分化受到抑制，且Smad1/5/9磷酸化水平减弱。上述结果表明，miR-765能够靶向作用BMP6经Smad1/5/9信号通路调控成骨分化。该项目所取得的系列成果将为丰富老年性骨质疏松发病机制和寻找新的治疗靶点提供依据。