BACKGROUND Some microRNAs (miRNAs) are involved in osteogenic differentiation. In recent years, increasing evidences have revealed that exosomes contain specific miRNAs. However, the effect and mechanism of miR-23a-5p-containing exosomes in osteoblast remain largely unclear. METHODS We extracted exosomes from RANKL-induced RAW 264.7 cells, and identified exosomes via transmission electron microscopy, western blot and flow cytometry analysis. In addition, exosome secretion was inhibited by GW4869 and Rab27a siRNAs. miR-23a-5p expression was analyzed by qRT-PCR, and the related protein levels were examined by western blot assay. Furthermore, the number and distribution of osteoclasts were detected by TRAP staining, and early osteogenesis was evaluated by ALP staining. Combination of YAP1 and Runx2 was verified by Co-IP assay, and the regulation of miR-23a-5p and Runx2 was measured by dual luciferase reporter assay. RESULTS We successfully extracted exosomes from RANKL-induced RAW 264.7 cells, and successfully verified exosomes morphology. We also indicated that miR-23a-5p was highly expressed in exosomes from RANKL-induced RAW 264.7 cells, and osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity, while its inhibition weakened osteoclasts. In mechanism, we demonstrated that Runx2 was a target gene of miR-23a-5p, YAP interacted with Runx2, and YAP or Runx2 inhibited MT1DP expression. In addition, we proved that knockdown of MT1DP facilitated osteogenic differentiation by regulating FoxA1 and Runx2. CONCLUSIONS We demonstrated that osteoclast-derived miR-23a-5p-containing exosomes could efficiently suppress osteogenic differentiation by inhibiting Runx2 and promoting YAP1-mediated MT1DP. Therefore, we suggested miR-23a-5p in exosomes might provide a novel mechanism for osteoblast function.

中文翻译：

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破骨细胞衍生的含有 miR-23a-5p 的外泌体通过调节 Runx2 抑制成骨分化。

背景一些微小RNA（miRNA）参与成骨分化。近年来，越来越多的证据表明外泌体含有特定的 miRNA。然而，含 miR-23a-5p 的外泌体在成骨细胞中的作用和机制仍不清楚。方法 我们从 RANKL 诱导的 RAW 264.7 细胞中提取外泌体，并通过透射电子显微镜、蛋白质印迹和流式细胞术分析鉴定外泌体。此外，外泌体分泌受到 GW4869 和 Rab27a siRNA 的抑制。通过qRT-PCR分析miR-23a-5p表达，通过蛋白质印迹法检测相关蛋白水平。此外，通过TRAP染色检测破骨细胞的数量和分布，通过ALP染色评估早期成骨。YAP1 和 Runx2 的组合通过 Co-IP 测定验证，并且通过双荧光素酶报告基因测定测量miR-23a-5p和Runx2的调节。结果我们成功地从RANKL诱导的RAW 264.7细胞中提取了外泌体，并成功验证了外泌体的形态。我们还表明 miR-23a-5p 在来自 RANKL 诱导的 RAW 264.7 细胞的外泌体中高表达，并且破骨细胞衍生的含有 miR-23a-5p 的外泌体抑制成骨细胞活性，而其抑制作用减弱了破骨细胞。在机制上，我们证明Runx2是miR-23a-5p的靶基因，YAP与Runx2相互作用，YAP或Runx2抑制MT1DP的表达。此外，我们证明敲低 MT1DP 通过调节 FoxA1 和 Runx2 促进成骨分化。结论 我们证明破骨细胞衍生的含有 miR-23a-5p 的外泌体可以通过抑制 Runx2 和促进 YAP1 介导的 MT1DP 有效抑制成骨分化。因此，我们认为外泌体中的 miR-23a-5p 可能为成骨细胞功能提供了一种新的机制。