Growth disorders in the orofacial bone development process may lead to orofacial deformities. The balance between bone matrix formation by mesenchymal lineage osteoblasts and bone resorption by osteoclasts is vital for orofacial bone development. Although the mechanisms of orofacial mesenchymal stem cells (OMSCs) in orofacial bone development have been studied intensively, the communication between OMSCs and osteoclasts remains largely unclear./nMethods: We used a neural crest cell-specific knockout mouse model to investigate orofacial bone development in GATA-binding protein 4 (GATA4) morphants. We investigated the underlying mechanisms of OMSCs-derived exosomes (OMExos) on osteoclastogenesis and bone resorption activity in vitro. miRNAs were extracted from OMExos, and differences in miRNA abundances were determined using an Affymetrix miRNA array. Luciferase reporter assays were used to validate the binding between GATA4 and miR-206-3p in OMSCs and to confirm the putative binding of miR-206-3p and its target genes in OMSCs and osteoclasts. The regulatory mechanism of the GATA4-miR-206-3p axis in OMSC osteogenic differentiation and osteoclastogenesis was examined in vitro and in vivo./nResults: Wnt1-Cre;Gata4^fl/fl mice (cKO) not only presented inhibited bone formation but also showed active bone resorption. Osteoclasts cocultured in vitro with cKO OMSCs presented an increased capacity for osteoclastogenesis, which was exosome-dependent. Affymetrix miRNA array analysis showed that miR-206-3p was downregulated in exosomes from shGATA4 OMSCs. Moreover, the transcriptional activity of miR-206-3p was directly regulated by GATA4 in OMSCs. We further demonstrated that miR-206-3p played a key role in the regulation of orofacial bone development by directly targeting bone morphogenetic protein-3 (Bmp3) and nuclear factor of activated T -cells, cytoplasmic 1 (NFATc1). OMExos and agomiR-206-3p enhanced bone mass in Wnt1-cre;Gata4^fl/fl mice by augmenting trabecular bone structure and decreasing osteoclast numbers./nConclusion: Our findings confirm that miR-206-3p is an important downstream factor of GATA4 that regulates the functions of OMSCs and osteoclasts. These results demonstrate the efficiency of OMExos and microRNA agomirs in promoting bone regeneration, which provide an ideal therapeutic tool for orofacial bone deformities in the future./n

中文翻译：

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GATA4 驱动的 miR-206-3p 特征通过调节成骨和破骨细胞活性来控制口面骨发育

口面部骨骼发育过程中的生长障碍可能导致口面部畸形。间充质成骨细胞的骨基质形成和破骨细胞的骨吸收之间的平衡对于口面骨的发育至关重要。尽管已经深入研究了口面间充质干细胞（OMSCs）在口面骨发育中的机制，但 OMSCs 与破骨细胞之间的通讯仍不清楚。/n方法：我们使用神经嵴细胞特异性敲除小鼠模型来研究口面骨发育在 GATA 结合蛋白 4 (GATA4) morphant 中。我们研究了 OMSCs 衍生的外泌体 (OMExos)在体外对破骨细胞生成和骨吸收活性的潜在机制. 从 OMExos 中提取 miRNA，并使用 Affymetrix miRNA 阵列确定 miRNA 丰度的差异。荧光素酶报告基因检测用于验证 OMSCs 中 GATA4 和 miR-206-3p 之间的结合，并确认 miR-206-3p 及其靶基因在 OMSCs 和破骨细胞中的推定结合。在体外和体内检测了 GATA4-miR-206-3p 轴在 OMSC 成骨分化和破骨细胞生成中的调节机制。/n结果： Wnt1-Cre；Gata4 ^fl/fl小鼠 (cKO) 不仅抑制骨形成，而且也表现出活跃的骨吸收。体外共培养的破骨细胞使用 cKO OMSCs 的破骨细胞生成能力增加，这是外泌体依赖性的。Affymetrix miRNA 阵列分析显示 miR-206-3p 在来自 shGATA4 OMSCs 的外泌体中下调。此外，miR-206-3p 的转录活性受 GATA4 在 OMSCs 中的直接调节。我们进一步证明了 miR-206-3p 通过直接靶向骨形态发生蛋白 3 (Bmp3) 和活化 T 细胞的核因子细胞质 1 (NFATc1) 在调节口面骨发育中发挥关键作用。OMExos 和 agomiR-206-3p通过增加骨小梁结构和减少破骨细胞数量来增强Wnt1-cre;Gata4 ^fl/fl 小鼠的骨量。/n结论：我们的研究结果证实，miR-206-3p 是 GATA4 的重要下游因子，可调节 OMSCs 和破骨细胞的功能。这些结果证明了 OMExos 和 microRNA agomirs 在促进骨再生方面的效率，为未来口面骨畸形提供了理想的治疗工具。/n