Department of Endodontics, School of Stomatology, China Medical University, Shenyang, 110002, P.R. China.
J Cell Biochem. 2018 Jan;119(1):536-546. doi: 10.1002/jcb.26212. Epub 2017 Jul 24.
This study aims to elucidate the mechanisms by which microRNA-143-5p (miR-143-5p) targets runt-related transcription factor 2 (Runx2) in the differentiation of dental pulp stem cells (DPSCs) into odontoblasts, through regulating the osteoprotegerin receptor activator of the nuclear factor-κB ligand (OPG/RANKL) signaling pathway. Following transfection, DPSCs were divided into blank, control, miR-143-5p mimics, miR-143-5p inhibitors, miR-143-5p inhibitors + siRunx2 and siRunx2 groups. Alkaline phosphatase (ALP) activity and mineralized nodules were detected using ALP kit and alizarin red staining. Quantitative reverse transcriptase real time PCR (qRT-PCR) was conducted to measure mRNA expressions of miR-143-5p, Runx2, OPG, and RANKL. Western blotting was used to assess protein expression of odontoblast differentiation-related proteins. Transwell assay and an extracellular matrix (ECM) adhesion cell assay were employed to examine cell migration and cell adhesion. Compared with the blank group, the miR-143-5p mimics and siRunx2 groups showed decreased ALP activity, decreased mineralized nodules and displays of calcium. Fewer migrated cells, weakened cell adhesion, decreased protein expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein 1 (DMP1), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), OPG and Runx2, and increased RANKL protein expressions were observed. Additionally, opposite results were observed in the miR-143-5p inhibitors group, demonstrating that down-regulated miR-143-5p promotes the differentiation of DPSCs into odontoblasts by enhancing Runx2 expression via the OPG/RANKL signaling pathway. Based on findings in this study, it is postulated that the enhancement of Runx2 expression via the regulation of the OPG/RANKL signaling pathway could be a beneficial approach for dental pulp regeneration. J. Cell. Biochem. 119: 536-546, 2018. © 2017 Wiley Periodicals, Inc.
本研究旨在阐明 microRNA-143-5p（miR-143-5p）通过调节核因子-κB 配体受体激活剂的骨保护素（OPG/RANKL）信号通路靶向 Runt 相关转录因子 2（Runx2）在牙髓干细胞（DPSCs）向成牙本质细胞分化中的作用机制。转染后，将 DPSCs分为空白组、对照组、miR-143-5p 模拟物组、miR-143-5p 抑制剂组、miR-143-5p 抑制剂+siRunx2 组和 siRunx2 组。使用碱性磷酸酶（ALP）试剂盒和茜素红染色检测 ALP 活性和矿化结节。采用实时定量逆转录 PCR（qRT-PCR）检测 miR-143-5p、Runx2、OPG 和 RANKL 的 mRNA 表达。Western blot 检测牙本质分化相关蛋白的表达。Transwell 试验和细胞外基质（ECM）黏附细胞试验检测细胞迁移和细胞黏附。与空白组相比，miR-143-5p 模拟物组和 siRunx2 组的 ALP 活性降低，矿化结节和钙显示减少。迁移细胞减少，细胞黏附减弱，牙本质磷蛋白（DPP）、牙本质涎蛋白（DSP）、牙本质基质蛋白 1（DMP1）、骨桥蛋白（OPN）、骨唾液蛋白（BSP）、骨钙素（OCN）、OPG 和 Runx2 的蛋白表达减少，RANKL 蛋白表达增加。miR-143-5p 抑制剂组则相反，结果表明下调 miR-143-5p 通过增强 OPG/RANKL 信号通路中的 Runx2 表达促进 DPSCs 向成牙本质细胞分化。基于本研究的结果，我们推测通过调节 OPG/RANKL 信号通路增强 Runx2 表达可能是牙髓再生的有益方法。J. Cell. Biochem. 119: 536-546, 2018. © 2017 Wiley Periodicals, Inc.
