Human mesenchymal stem cells (hMSCs) are multipotent cells that can be differentiated into different cell types including osteoblasts. Herein we aimed to assess the regulation of transcription factor mesenchyme homeobox 1 (Meox1) in the osteogenic differentiation of hMSCs and to determine the microRNA which targets on Meox1. Total RNA was extracted from the isolated ligamentum flavum tissue samples and cultured hMSCs, and the expression of Meox1 was assessed by RT-PCR and Western blot assays. Cultured hMSCs were induced towards osteoblastic differentiation, and the osteoblast phenotype was determined by alkaline phosphatase activity and alizarin red staining. The microRNA targeting on the 3′-UTR of Meox1was predicted using bioinformatics tool, and the binding was validated by luciferase and RNA pulldown assays. The osteoblastic differentiation of hMSCs was checked with the knockdown of Meox1 and microRNA inhibitors. Higher expression of Meox1, and lower expression of miR-3064–5p in ossified ligamentum flavum (OLF) tissues were identified. In addition, increased expression along with the osteoblastic differentiation of hMSCs was found. Further research revealed that Meox was a direct target of miR-3064–5p, when the former promoted the differentiation of hMSCs into osteoblasts, the latter significantly suppressed the osteogenesis. The expression of Meox1 increased gradually with the osteoblastic differentiation of hMSCs, during which miR-3064–5p decreased. Meox1 is a direct target of miR-3064–5p, and they both play important roles in the osteogenesis. These findings provide potential target for the development of therapeutic drugs for skeletal system diseases.

人间充质干细胞 (hMSCs) 是多能细胞，可以分化成不同的细胞类型，包括成骨细胞。在这里，我们旨在评估转录因子间充质同源框 1 (Meox1) 在 hMSCs 成骨分化中的调节作用，并确定靶向 Meox1 的 microRNA。从分离的黄韧带组织样品和培养的 hMSCs 中提取总 RNA，并通过 RT-PCR 和蛋白质印迹分析评估 Meox1 的表达。培养的hMSCs诱导成骨细胞分化，通过碱性磷酸酶活性和茜素红染色确定成骨细胞表型。使用生物信息学工具预测了靶向 Meox1 的 3'-UTR 的 microRNA，并通过荧光素酶和 RNA pulldown 测定验证了结合。通过敲低 Meox1 和 microRNA 抑制剂检查 hMSCs 的成骨细胞分化。确定了骨化黄韧带（OLF）组织中 Meox1 的较高表达和 miR-3064-5p 的较低表达。此外，发现hMSCs的表达增加以及成骨细胞分化。进一步的研究表明，Meox 是 miR-3064-5p 的直接靶点，当前者促进 hMSCs 向成骨细胞分化时，后者显着抑制成骨。随着hMSCs成骨细胞分化，Meox1的表达逐渐增加，在此期间miR-3064-5p下降。Meox1 是 miR-3064-5p 的直接靶标，它们都在成骨过程中发挥重要作用。