State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
Department of Conservative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
Int Endod J. 2024 Nov;57(11):1623-1638. doi: 10.1111/iej.14120. Epub 2024 Jul 7.
The regenerative capacity of dental pulp relies on the odonto/osteogenic differentiation of dental pulp cells (DPCs), but dynamic microenvironmental changes hinder the process. Bone morphogenetic protein 9 (BMP9) promotes differentiation of DPCs towards an odonto/osteogenic lineage, forming dentinal-like tissue. However, the molecular mechanism underlying its action remains unclear. This study investigates the role of DLX6 antisense RNA 1 (DLX6-AS1) in odonto/osteogenic differentiation induced by BMP9.
Custom RT profiler PCR array, quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of DLX6-AS1 and its potential signal axis. Osteogenic ability was evaluated using alkaline phosphatase and alizarin red S staining. Interactions between lncRNA and miRNA, as well as miRNA and mRNA, were predicted through bioinformatic assays, which were subsequently validated via RNA immunoprecipitation and dual luciferase reporter assays. Student's t-test or one-way ANOVA with post hoc Tukey HSD tests were employed for data analysis, with a p-value of less than .05 considered statistically significant.
DLX6-AS1 was upregulated upon BMP9 overexpression in DPCs, thereby promoting odonto/osteogenic differentiation. Additionally, miR-128-3p participated in BMP9-induced odonto/osteogenic differentiation by interacting with the downstream signal MAPK14. Modifying the expression of miR-128-3p and transfecting pcMAPK14/siMAPK14 had a rescue impact on odonto/osteogenic differentiation downstream of DLX6-AS1. Lastly, miR-128-3p directly interacted with both MAPK14 and DLX6-AS1.
DLX6-AS1 could regulate the odonto/osteogenic differentiation of DPCs under the control of BMP9 through the miR-128-3p/MAPK14 axis.
牙髓的再生能力依赖于牙髓细胞（DPCs）的牙源性/成骨分化，但动态微环境变化会阻碍这一过程。骨形态发生蛋白 9（BMP9）促进 DPCs 向牙源性/成骨谱系分化，形成类似牙本质的组织。然而，其作用的分子机制尚不清楚。本研究探讨了反义 RNA 1（DLX6-AS1）在 BMP9 诱导的牙源性/成骨分化中的作用。
采用定制 RT 谱 PCR 阵列、定量实时 PCR（qRT-PCR）和 Western blot 检测 DLX6-AS1 的表达模式及其潜在信号轴。通过碱性磷酸酶和茜素红 S 染色评估成骨能力。通过生物信息学分析预测 lncRNA 和 miRNA 以及 miRNA 和 mRNA 之间的相互作用，随后通过 RNA 免疫沉淀和双荧光素酶报告基因检测进行验证。采用 Student's t-test 或单因素方差分析（one-way ANOVA）加事后 Tukey HSD 检验进行数据分析，p 值小于 0.05 为差异有统计学意义。
在 DPCs 中转染 BMP9 可上调 DLX6-AS1 的表达，从而促进牙源性/成骨分化。此外，miR-128-3p 通过与下游信号 MAPK14 相互作用参与 BMP9 诱导的牙源性/成骨分化。改变 miR-128-3p 的表达并转染 pcMAPK14/siMAPK14 对 DLX6-AS1 下游的牙源性/成骨分化有挽救作用。最后，miR-128-3p 直接与 MAPK14 和 DLX6-AS1 相互作用。
DLX6-AS1 可通过 miR-128-3p/MAPK14 轴调控 BMP9 控制下的 DPCs 牙源性/成骨分化。
