Liao Xianmin, Yang Zhenjin, Li Yan, Cui Yun, Ma Liya, Liang Cun, Guan Zheng, Hu Jiangtian
Department of Stomatology Centre, The First People's Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, Kunming, 650032, Yunnan, China.
Department of Orthodontics, Kunming Medical University Affiliated Stomatological Hospital, Building C, Hecheng International, No. 1088 Middle Haiyuan Road, Kunming 650106, Yunnan, China.
Free Radic Biol Med. 2025 Sep;237:88-100. doi: 10.1016/j.freeradbiomed.2025.05.425. Epub 2025 May 29.
Our previous findings revealed that exosomes derived from M2-polarized macrophages enhance the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), and identified key microRNAs (miRNAs) using high-throughput miRNA sequencing. Therefore, the present study aimed to elucidate the role and underlying molecular mechanism by which exosomes derived from M2 macrophages mediate the osteogenic differentiation of hPDLSCs.
Following lentiviral-mediated modulation of miR-6879-5p in both hPDLSCs and M2 macrophage-derived exosomes, RT-qPCR, western blotting, and Alizarin Red staining were applied to assess alterations in osteogenic markers, including ALP, OCN, Collagen I, and RUNX2, as well as mineralized nodule formation in hPDLSCs. Immunoprecipitation-mass spectrometry (IP-MS) was employed to identify proteins interacting with miR-6879-5p target genes in hPDLSCs.
Knockdown of miR-6879-5p in the exosomes reduced the expression of osteogenic markers and inhibited calcified nodule formation in hPDLSCs. Overexpression of TRIM26 attenuated the osteogenic differentiation of hPDLSCs, an effect that was reversed by miR-6879-5p overexpression. IP-MS identified 410 TRIM26-interacting proteins in hPDLSCs. These proteins were associated with ubiquitination, aerobic glycolysis, and amino acid metabolism. The hub proteins in the TRIM26-associated PPI network included RPL and RPS family proteins, as well as glycolysis-associated proteins. CO-IP confirmed an interaction between TRIM26 and PKM, and showed that TRIM26 increased PKM ubiquitination. Overexpression of PKM rescued TRIM26-mediated suppression of osteogenic marker expression and mineralized nodule formation in hPDLSCs.
miR-6879-5p carried by M2 macrophage-derived exosomes promotes osteogenic differentiation and aerobic glycolysis in hPDLSCs via modulating TRIM26-mediated ubiquitination of PKM.
我们之前的研究结果表明，源自M2极化巨噬细胞的外泌体可增强人牙周膜干细胞（hPDLSCs）的成骨分化，并通过高通量miRNA测序鉴定了关键的微小RNA（miRNA）。因此，本研究旨在阐明M2巨噬细胞来源的外泌体介导hPDLSCs成骨分化的作用及潜在分子机制。
在hPDLSCs和M2巨噬细胞来源的外泌体中通过慢病毒介导对miR-6879-5p进行调控后，应用逆转录-定量聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法和茜素红染色来评估hPDLSCs中成骨标志物（包括碱性磷酸酶（ALP）、骨钙素（OCN）、I型胶原蛋白和RUNX2）的变化以及矿化结节的形成。采用免疫沉淀-质谱法（IP-MS）鉴定hPDLSCs中与miR-6879-5p靶基因相互作用的蛋白质。
外泌体中miR-6879-5p的敲低降低了hPDLSCs中成骨标志物的表达并抑制了钙化结节的形成。TRIM26的过表达减弱了hPDLSCs的成骨分化，而miR-6879-5p的过表达可逆转这一效应。IP-MS在hPDLSCs中鉴定出410种与TRIM26相互作用的蛋白质。这些蛋白质与泛素化、有氧糖酵解和氨基酸代谢相关。TRIM26相关蛋白质-蛋白质相互作用（PPI）网络中的枢纽蛋白包括核糖体蛋白L（RPL）和核糖体蛋白S（RPS）家族蛋白以及糖酵解相关蛋白。免疫共沉淀（CO-IP）证实了TRIM26与丙酮酸激酶M2（PKM）之间的相互作用，并表明TRIM26增加了PKM的泛素化。PKM的过表达挽救了TRIM26介导的hPDLSCs中成骨标志物表达和矿化结节形成的抑制作用。
M2巨噬细胞来源的外泌体携带的miR-6879-5p通过调节TRIM26介导的PKM泛素化促进hPDLSCs的成骨分化和有氧糖酵解。
