LncRNA DLX6-AS1介导BMP9/MAPK信号轴调控牙髓干细胞成牙本质分化的机制研究

The health of dental pulp which plays a crucial role in the function of teeth directly influences the lifetime of teeth. When the dental pulp is exposed to injury, the self-reparative mechanism will be initiated to protect the dental pulp from exposures. The key to this mechanism is the odontoblastic differentiation of dental pulp stem cells (DPSCs). Bone morphogenetic protein 9 (BMP9) is one of most osteogenic BMPs among the human BMP family, which plays an important role in the differentiation of a variety of stem cells. Our previous study found that BMP9 may induce osteo/odontoblastic differentiation of DPSCs and activate MAPK signaling pathways. Whole transcriptome sequencing under BMP9 activation revealed high expression of LncRNA DLX6-AS1, suggesting that BMP9 may effectively promote the repair of dental pulp by regulating the differentiation of DPSCs. The mechanism behind it is still not fully understood. Combined with our previous studies, the purpose of this study are as follows: 1) confirm the expression variation of BMP9/MAPK signaling axis and LncRNA DLX6-AS1 in the different stages of pulp injury repair in dental pulp tissues; 2) explore the function and mechanism of LncRNA DLX6-AS1 in the DPSCs differentiation regulated by BMP9/MAPK signaling axis through applying techniques such as cytoplasmic/nuclear RNA isolation with qRT-PCR assay, RNA scope-ISH, RNA Immunoprecipitation; 3) verify the function of BMP9 in repairing the injured pulp using a conditioned overexpressing mice model. Therefore, the results of this project are expected to further expand the regulation mechanism of dental pulp stem cell differentiation and provide a new theoretical perspective and data support for improving the clinical effectiveness of pulp repair mechanism.

牙髓损伤后会启动自我修复机制以保护牙髓，牙髓干细胞(DPSCs)的成牙本质分化是这一机制的关键。BMP9是诱导干细胞成骨分化最有效的BMPs，在多种干细胞分化中发挥重要作用。申请人前期研究发现BMP9可诱导DPSCs成牙本质分化，促进LncRNA DLX6-AS1特异性高表达，提示BMP9有促进牙髓损伤修复的潜能，其作用机制尚不清楚。结合前期工作，本项目拟首先明确BMP9/MAPK信号轴、LncRNA DLX6-AS1在牙髓损伤修复不同阶段的b表达变化；其次通过核质分离、RNAscope-ISH、RIP等技术深入探究LncRNA DLX6-AS1介导BMP9/MAPK信号轴调控DPSCs分化的作用机制；最后运用条件性基因过表达小鼠对BMP9促进牙髓损伤修复进行功能验证。本项目研究成果有望进一步拓展牙髓干细胞分化的调控机制，为提升牙髓修复机制的临床效用提供新的理论视角及数据支持。

牙髓损伤后会启动自我修复机制以保护牙髓，牙髓细胞(DPCs)的成牙本质分化是这一机制的关键。BMP9是诱导干细胞成骨分化最有效的BMPs，在多种干细胞分化中发挥重要作用。课题组前期研究发现BMP9可以诱导DPCs成牙本质分化，同时伴LncRNA DLX6-AS1高表达，但具体机制有待厘清。本项目首先明确BMP9/MAPK信号轴、DLX6-AS1在牙髓损伤修复不同阶段的表达变化；随后探究DLX6-AS1介导BMP9/MAPK信号轴调控DPCs分化的作用机制；最后对BMP9促进牙髓损伤修复进行功能验证。.研究发现：.1. DLX6-AS1的表达在BMP9诱导后上调、在siRNA干扰后下调。在BMP9诱导后敲低DLX6-AS1可以导致早期和晚期成牙本质分化标志基因的表达回落。将BMP9诱导后的牙本质块-牙髓细胞复合物植入裸鼠皮下后显著表达DSPP、DMP1并生成牙本质样组织。.2. 牙髓损伤修复中炎症相关的关键基因的过表达可能抑制牙髓成牙本质分化，miR-128-3p–MAPK14信号轴可能为DLX6-AS1的下游作用靶点。.3. DLX6-AS1–miR-128-3p–MAPK14间可能存在以竞争性内源RNA为机制的分子交互。RNA免疫共沉淀实验发现Ago2蛋白可以富集DLX6-AS1、miR-128-3p、MAPK14。荧光素酶报告实验显示DLX6-AS1可以同miR-128-3p直接结合。敲低DLX6-AS1可以导致p38总蛋白p-p38蛋白表达下降。过表达BMP9时加入miR-128-3p mimics可以降低ALP、RUNX、DMP1和DSPP的表达，而再加入rh p38α可使上述表达回复；敲低DLX6-AS1可以使miR-128-3p mimics上调，MAPK14、ALP、RUNX、DMP1和DSPP下调，在此时反向调控miR-128-3p和MAPK14的蛋白产物p38α可以使上述表达回复。.DLX6-AS1是 BMP9诱导DPCs成牙本质分化过程中的关键信号分子。牙髓损伤修复激活了大量免疫细胞和炎症通路，DLX6-AS1–miR-128-3p–MAPK14通路可以通过竞争性内源RNA机制参与BMP9诱导的DPCs成牙本质分化。本项目研究成果有望进一步拓展牙髓细胞分化的调控机制，为提升牙髓修复机制的临床效用提供新的理论视角及数据支持。