Department of Orthopedics, the First Affiliated Hospital of Kunming Medical University, Kunming, China.
FEBS Open Bio. 2020 Nov;10(11):2499-2506. doi: 10.1002/2211-5463.12979. Epub 2020 Oct 25.
Osteoporosis and osteoarthritis are orthopedic disorders that affect millions of elderly people worldwide; stimulation of bone formation is a potential therapeutic strategy for the treatment of these conditions. As the only bone-forming cells, osteoblasts play a key role in bone reconstruction. The microRNA miR-17-3p is downregulated during osteogenic differentiation of human bone marrow mesenchymal stem cells, but its precise role in this process is unknown. Here, we investigated the role of miR-17-3p in osteoblast differentiation. An in vitro model of osteogenesis was established by treating MC3T3-E1 murine preosteoblast cells with bone morphogenetic protein 2 (BMP2). The expression of miR-17-3p in BMP2-induced MC3T3-E1 cells was detected by reverse transcription-quantitative PCR, and its effects on cells transfected with miR-17-3p mimic or inhibitor were evaluated by Alizarin Red staining, alkaline phosphatase (ALP) activity assay, and by detection of osteoblast markers including the ALP, collagen type I α1 chain, and osteopontin genes. Bioinformatics analysis was carried out to identify putative target genes of miR-17-3p, and the luciferase reporter assay was used for functional validation. Rescue experiments were performed to determine whether SRY-box transcription factor 6 (Sox6) plays a role in the regulation of osteoblast differentiation by miR-17-3p. We report that miR-17-3p was downregulated upon BMP2-induced osteoblast differentiation in MC3T3-E1 cells, and this was accompanied by decreased differentiation and mineralization, ALP activity, and expression of osteogenesis-related genes. Sox6 was confirmed to be a target gene of miR-17-3p in osteoblasts, and the inhibitory effect of miR-17-3p on osteoblast differentiation was observed to occur via Sox6. These results suggest the existence of a novel mechanism underlying miRNA-mediated regulation of osteogenesis, which has potential implications for the treatment of orthopedic disorders.
骨质疏松症和骨关节炎是影响全球数百万老年人的骨科疾病；刺激骨形成是治疗这些疾病的潜在治疗策略。成骨细胞作为唯一的成骨细胞，在骨重建中发挥关键作用。微小 RNA miR-17-3p 在人骨髓间充质干细胞成骨分化过程中下调，但在这一过程中的精确作用尚不清楚。在这里，我们研究了 miR-17-3p 在成骨细胞分化中的作用。通过用骨形态发生蛋白 2 (BMP2) 处理 MC3T3-E1 鼠前成骨细胞建立体外成骨模型。通过逆转录定量 PCR 检测 BMP2 诱导的 MC3T3-E1 细胞中 miR-17-3p 的表达，并通过茜素红染色、碱性磷酸酶 (ALP) 活性测定以及检测成骨细胞标志物，包括 ALP、胶原 I α1 链和骨桥蛋白基因，评估转染 miR-17-3p 模拟物或抑制剂对细胞的影响。进行了生物信息学分析以鉴定 miR-17-3p 的潜在靶基因，并使用荧光素酶报告基因检测进行功能验证。进行了挽救实验以确定 SRY 盒转录因子 6 (Sox6) 是否在 miR-17-3p 调节成骨细胞分化中发挥作用。我们报告说，在 MC3T3-E1 细胞中，BMP2 诱导的成骨分化导致 miR-17-3p 下调，同时伴随着分化和矿化减少、ALP 活性和骨形成相关基因表达降低。Sox6 被确认为成骨细胞中 miR-17-3p 的靶基因，并且观察到 miR-17-3p 对成骨细胞分化的抑制作用是通过 Sox6 发生的。这些结果表明，miRNA 介导的成骨调控存在一种新的机制，这对于治疗骨科疾病具有潜在意义。
