Table1_miR-330–5p in Small Extracellular Vesicles Derived From Plastrum testudinis-Preconditioned Bone Mesenchymal Stem Cells Attenuates Osteogenesis by Modulating Wnt/β-Catenin Signaling.docx

The bone microenvironment is crucial for the growth and development of different types of osteocytes. Small extracellular vesicles (sEVs) secreted by bone mesenchymal stem cells are delivered to target cells where their contents regulate biological functions. Here, we evaluated the osteogenic effects and mechanism of sEVs derived from Plastrum testudinis-preconditioned bone mesenchymal stem cells (PT-sEV). The osteogenic effects of PT-sEV were evaluated by the differentiation of osteoblasts and the alternation of bone quality and quantity in ovariectomized rats. The specific mechanism was explored by high-throughput sequencing and verified by transfection with the corresponding miRNA mimic and inhibitor. RNA-sequence identified a unique enrichment of a set of miRNAs in PT-sEV compared with sEVs derived from untreated BMSCs. Overexpression or inhibition in vitro indicated that the osteogenic inducing potential of sEVs was mainly attributable to miR-330-5p, one of the most dramatically downregulated miRNAs in the PT-sEV fraction. Dual luciferase reporter assays showed that miR-330-5p negatively regulated osteogenesis by directly binding to the 3′ untranslated region of Tnc. Additional experiments showed that Tnc regulated Wnt/β-catenin signaling, and rescue experiment showed that miR-330-5p could restore β-catenin expression; additionally, animal experiments indicated that Wnt signaling was inactivated in the ovariectomized rats. These data demonstrated the regenerative potential of PT-sEV, which induced osteogenic differentiation of pre-osteoblasts, leading to bone formation. This process was achieved by delivering miR-330-5p, which regulated Tnc to control Wnt/β-catenin signaling.

骨微环境对于不同类型骨细胞的生长与发育至关重要。由骨髓间充质干细胞（bone mesenchymal stem cells, BMSCs）分泌的小细胞外囊泡（small extracellular vesicles, sEVs）可被递送至靶细胞，其内含物能够调控靶细胞的生物学功能。本研究评估了经龟板（Plastrum testudinis）预处理的骨髓间充质干细胞来源的sEVs（PT-sEV）的成骨效应及其作用机制。通过成骨细胞分化水平以及去卵巢大鼠的骨质量与骨量变化，对PT-sEV的成骨效应进行评价。采用高通量测序探索其具体作用机制，并通过转染对应miRNA模拟物与抑制剂开展验证实验。RNA测序结果显示，与未处理的BMSCs来源的sEVs相比，PT-sEV中存在一组miRNA的特异性富集。体外过表达或抑制实验表明，sEVs的成骨诱导潜能主要源于miR-330-5p——该分子是PT-sEV组分中下调幅度最为显著的miRNA之一。双荧光素酶报告基因实验证实，miR-330-5p可通过直接结合Tnc的3'非翻译区，负向调控成骨过程。额外实验显示，Tnc可调控Wnt/β-连环蛋白（Wnt/β-catenin）信号通路；拯救实验表明，miR-330-5p能够恢复β-连环蛋白的表达；此外动物实验结果显示，去卵巢大鼠体内的Wnt信号通路处于失活状态。本研究数据证实了PT-sEV的再生潜能：其可诱导前成骨细胞发生成骨分化，进而促进骨形成。这一过程通过递送miR-330-5p实现，该miRNA通过调控Tnc以控制Wnt/β-连环蛋白信号通路。