Bone marrow mesenchymal stem cells (BMSCs) exhibit the capacity to self-renew and differentiate into multi-lineage cell types, including osteoblasts, which are crucial regulators of fracture healing. Thus, this study aims to investigate the effect of microRNA (miR)-22-3p from BMSC-derived EVs on osteogenic differentiation and its underlying mechanism. Extracellular vesicles (EVs) were isolated from BMSCs and taken up with BMSCs. Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-22-3p and FTO. Loss- and gain-of-function experiments were performed to determine the roles of EV-delivered miR-22-3p and FTO in osteogenic differentiation as well as their regulatory role in the MYC/PI3K/AKT axis. To determine the osteogenic differentiation, ALP and ARS stainings were conducted, and the levels of RUNX2, OCN, and OPN level were determined. In vivo experiment was conducted to determine the function of EV-delivered miR-22-3p and FTO in osteogenic differentiation, followed by ALP and ARS staining. miR-22-3p expression was repressed, while FTO expression was elevated in the ovariectomized mouse model. Overexpression of miR-22-3p, EV-delivered miR-22-3p, increased ALP activity and matrix mineralization of BMSCs and promoted RUNX2, OCN, and OPN expressions in BMSCs. miR-22-3p negatively targeted FTO expression. FTO silencing rescued the suppressed osteogenic differentiation by EV-delivered miR-22-3p inhibitor. FTO repression inactivated the MYC/PI3K/AKT pathway, thereby enhancing osteogenic differentiation both in vivo and in vitro. In summary, miR-22-3p delivered by BMSC-derived EVs could result in the inhibition of the MYC/PI3K/AKT pathway, thereby promoting osteogenic differentiation via FTO repression.

中文翻译：

---

来自骨髓间充质干细胞的细胞外囊泡包裹的 miR-22-3p 通过 FTO 抑制促进成骨分化。

骨髓间充质干细胞 (BMSCs) 表现出自我更新和分化成多系细胞类型的能力，包括成骨细胞，它们是骨折愈合的关键调节剂。因此，本研究旨在研究来自 BMSC 衍生 EV 的 microRNA (miR)-22-3p 对成骨分化的影响及其潜在机制。细胞外囊泡 (EVs) 从 BMSCs 中分离出来并被 BMSCs 吸收。双荧光素酶报告基因检测用于验证 miR-22-3p 与 FTO 之间的结合关系。进行功能丧失和获得实验以确定 EV 传递的 miR-22-3p 和 FTO 在成骨分化中的作用以及它们在 MYC/PI3K/AKT 轴中的调节作用。为了确定成骨分化，进行了 ALP 和 ARS 染色，以及 RUNX2、OCN、和OPN水平确定。进行体内实验以确定 EV 传递的 miR-22-3p 和 FTO 在成骨分化中的功能，然后进行 ALP 和 ARS 染色。在卵巢切除小鼠模型中，miR-22-3p 表达被抑制，而 FTO 表达升高。miR-22-3p 的过表达，EV 传递的 miR-22-3p，增加了 BMSCs 的 ALP 活性和基质矿化，并促进了 BMSCs 中 RUNX2、OCN 和 OPN 的表达。miR-22-3p 负靶向 FTO 表达。FTO 沉默通过 EV 传递的 miR-22-3p 抑制剂挽救了抑制的成骨分化。FTO 抑制使 MYC/PI3K/AKT 通路失活，从而增强体内和体外的成骨分化。总之，由 BMSC 衍生的 EV 传递的 miR-22-3p 可能导致 MYC/PI3K/AKT 通路的抑制，