BACKGROUND The tight coupling between osteoblasts and osteoclasts is essential to maintain bone homeostasis. Deregulation of this process leads to loss and deterioration of the bone tissue causing diseases, such as osteoporosis. MicroRNAs are able to control cell differentiation of bone cells and thus, have been explored as therapeutic tools. In this study, we explored the potential of miR-99a-5p to concurrently modulate osteogenic differentiation, osteoclastogenesis, and the osteoblasts-osteoclasts crosstalk. METHODS To achieve this goal, human primary Mesenchymal Stem/Stromal Cells were differentiated into osteoblasts and adipocytes, and miR-99a-5p expression was evaluated by RT-qPCR. Knockdown and overexpression experiments were conducted to modulate miR-99a-5p expression in MC3T3 cells. Cell proliferation and cell death/apoptosis were evaluated by resazurin assay and flow cytometry, respectively. Proteomic analysis was used to identify the miR-99a-5p regulatory network, and ELISA to evaluate OPG levels in the cell culture supernatant. Conditioned media from MC3T3-transfected cells was incubated with RAW 264.7 cells and the effect on osteoclast differentiation was assessed. Human primary monocytes were isolated to induce osteoclastogenesis and evaluate miR-99a-5p expression. Finally, levels of miR-99a-5p were modulated in RAW 264.7 cells to understand the impact on osteoclastogenesis. RESULTS The results show that miR-99a-5p is significantly downregulated during the early stages of human primary MSCs osteogenic differentiation and during MC3T3 osteogenic differentiation. On the other hand, in hMSCs, miR-99a-5p levels are increased during the initial stages of adipogenic differentiation. Inhibition of miR-99a-5p in MC3T3 pre-osteoblastic cells promoted osteogenic differentiation, whereas its overexpression suppressed the levels of osteogenic specific genes (Runx2 and Alpl), as well as mineralization, with no effect on proliferation or apoptosis. Proteomic analysis of miR-99a-5p-transfected cells showed that numerous proteins known to be involved in cell differentiation were altered, including osteogenic differentiation markers and extracellular matrix proteins. While inhibition of miR-99a-5p increased the Tnfrsf11b (OPG encoding gene)/Tnfsf11 (RANKL encoding gene) mRNA expression ratio, in addition to increasing OPG secretion, miR-99a-5p overexpression resulted in the opposite effect. The cell culture supernatant of miR-99a-5p-inhibited MC3T3 cells impaired the osteoclastogenic potential of RAW 264.7 cells by decreasing the number of multinucleated cells and reducing the expression of osteoclastogenic markers. Interestingly, miR-99-5p expression is increased during osteoclasts differentiation, both in human primary monocytes and RAW 264.7. These results show that miR-99a-5p per se is a positive regulator of osteoclastogenic differentiation. CONCLUSIONS Globally, our findings show that miR-99a-5p inhibition simultaneously promotes the commitment into osteogenic differentiation, impairs osteoclastogenic differentiation, and control bone cells communication. Ultimately, it supports miR-99a-5p as a candidate for future novel miRNA-based therapies for bone diseases associated with bone remodeling deregulation.

中文翻译：

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miR-99a 在骨稳态中的作用：调节成骨谱系定向和破骨细胞分化

背景成骨细胞和破骨细胞之间的紧密耦合对于维持骨稳态是必不可少的。这个过程的失调导致骨组织的损失和恶化，从而导致疾病，例如骨质疏松症。MicroRNA 能够控制骨细胞的细胞分化，因此已被探索为治疗工具。在这项研究中，我们探索了 miR-99a-5p 同时调节成骨分化、破骨细胞生成和成骨细胞-破骨细胞串扰的潜力。方法为实现这一目标，人类原代间充质干/基质细胞分化为成骨细胞和脂肪细胞，并通过 RT-qPCR 评估 miR-99a-5p 表达。进行敲低和过表达实验以调节 MC3T3 细胞中的 miR-99a-5p 表达。分别通过刃天青测定和流式细胞术评估细胞增殖和细胞死亡/凋亡。蛋白质组学分析用于鉴定 miR-99a-5p 调节网络，ELISA 用于评估细胞培养上清液中的 OPG 水平。来自 MC3T3 转染细胞的条件培养基与 RAW 264.7 细胞一起孵育，并评估对破骨细胞分化的影响。分离人类原代单核细胞以诱导破骨细胞生成并评估 miR-99a-5p 表达。最后，在 RAW 264.7 细胞中调节 miR-99a-5p 的水平以了解对破骨细胞生成的影响。结果结果表明，在人原代MSCs成骨分化的早期阶段和MC3T3成骨分化过程中，miR-99a-5p显着下调。另一方面，在 hMSCs 中，miR-99a-5p 水平在成脂分化的初始阶段增加。在 MC3T3 前成骨细胞中抑制 miR-99a-5p 促进了成骨分化，而其过表达抑制了成骨特异性基因（Runx2 和 Alpl）的水平以及矿化，对增殖或凋亡没有影响。miR-99a-5p 转染细胞的蛋白质组学分析表明，许多已知参与细胞分化的蛋白质发生了改变，包括成骨分化标志物和细胞外基质蛋白。虽然 miR-99a-5p 的抑制增加了 Tnfrsf11b（OPG 编码基因）/Tnfsf11（RANKL 编码基因）mRNA 表达比率，但除了增加 OPG 分泌外，miR-99a-5p 过表达导致了相反的效果。miR-99a-5p 抑制的 MC3T3 细胞的细胞培养上清液通过减少多核细胞的数量和降低破骨细胞标记物的表达来损害 RAW 264.7 细胞的破骨细胞生成潜力。有趣的是，在破骨细胞分化过程中，人原代单核细胞和 RAW 264.7 中 miR-99-5p 的表达增加。这些结果表明 miR-99a-5p 本身是破骨细胞分化的正调节剂。结论在全球范围内，我们的研究结果表明 miR-99a-5p 抑制同时促进成骨分化的承诺，损害破骨细胞分化，并控制骨细胞通讯。最终，它支持 miR-99a-5p 作为未来新的基于 miRNA 的疗法的候选者，用于与骨重塑失调相关的骨病。