LncRNA MEG3介导BCL-2调控牙髓细胞防御修复的作用机制研究

Immune defense and reparative response were induced in dental pulp tissue in response to external stimuli. Recent studies have found that cell apoptosis was involved in the reparative response of dental pulp to external stimuli. However, the specific mechanism is unclear. In our preliminary studies, we applied the lncRNAs microarray technology to detect and screen the specific lncRNAs of inflamed dental pulp tissue, which was validated by the real-time PCR. We found that lncRNA MEG3 was significantly up-regulated in inflamed dental pulp tissue and LPS or MDP stimulated - dental pulp cells. Then we used RNA pull - down experiments to screen and found that the MEG3 combinds with the anti-apoptotic protein BCL-2. According to our results, we propose the possible mechanism that MEG3 may regulate the dental pulp cell apoptosis through combining with BCL-2 then mediate odontoblastic differentiation of dental pulp cells to participate in the dental pulp repair. Proposed on the basis of previous work, in this study project, we firstly intend to confirm whether lncRNA MEG3 participate in the regulation of dental pulp cell apoptosis. Then we will further analyze the combination zone of MEG3 and BCL-2 and discuss the possible interaction mechanism. Finally we will explore the effects of MEG3 mediated-apoptosis in dental pulp cells differentiation, to clarify the potential mechanism of lncRNA MEG3 in dental pulp reparative reaction and thus provide theoretical basis for in-depth research on the reparative response mechanism of dental pulp cells.

牙髓组织受外界刺激时产生免疫防御及修复反应。研究发现细胞凋亡参与牙髓组织对外界刺激的修复反应，但具体机制未明。本课题组前期研究应用lncRNAs芯片技术筛选出炎症牙髓特异表达的lncRNAs，发现参与细胞凋亡调控的lncRNA MEG3在炎症牙髓组织中显著高表达；RNA pull-down实验显示MEG3与抗凋亡蛋白BCL-2结合，据此我们提出MEG3参与调控牙髓防御修复的可能机制，即MEG3通过与BCL-2结合促进细胞凋亡，激活TGF-β信号通路促进牙髓细胞成牙本质向分化。本研究拟采用慢病毒转染、免疫共沉淀等方法进一步明确lncRNA MEG3是否参与调控牙髓细胞凋亡；分析MEG3与BCL-2的特异性结合区域及两者的相互作用机理；观察MEG3介导的牙髓细胞凋亡对牙髓细胞成牙本质向分化的影响，阐明lncRNA MEG3在牙髓损伤修复反应中的作用，为深入研究牙髓损伤修复机制提供新思路。

牙髓炎是因微生物入侵感染深层牙髓组织而产生的炎症，此时的组织修复依赖于具有多向分化潜能的人牙髓细胞（human dental pulp cells, hDPCs）进行成牙本质向分化。长链非编码RNA（Long noncoding RNAs, lncRNAs）可调控多种病理及生理过程，但其在牙髓炎症和修复过程中所起作用尚不明确。本研究采用高通量测序技术筛选了在人炎症牙髓和正常牙髓组织中差异表达的lncRNAs并明确了长链非编码RNA 母系印记基因3 (lncRNA maternally expressed gene 3, lncRNA MEG3)在人炎症牙髓组织和LPS诱导hDPCs中均表达升高。其中我们使用了RNAscope®技术来检测lncRNA MEG3在人牙髓组织的表达。通过RNA pulldown技术我们筛选了可能与lncRNA MEG3结合的蛋白及其潜在作用机制。通过建立lncRNA MEG3表达敲低模型我们探讨了lncRNA MEG3对LPS诱导hDPCs炎症因子产生以及其对hDPCs成牙本质向分化的作用。下调lncRNA MEG3可抑制LPS诱导hDPCs炎症因子TNF-α、IL-1β和 IL-6的表达，且该作用可能与p38/MAPK信号通路相关。同时下调lncRNA MEG3通过影响Wnt/β-catenin信号通路促进了hDPCs成牙本质向分化。我们结果提示lncRNA MEG3在牙髓炎中起促进炎症和抑制修复的作用。