Studies have proved that miRNAs participate in the regulation of osteoblast differentiation (OD), and abnormal expression of miRNAs is related with various states of OD. In this study, we investigated the role of miRNA-1-3p in OD using MC3T3-E1 cells. BMP2 is used to induce OD of MC3T3-E1 cells. MiRNA-1-3p mimics or miRNA-1-3p inhibitor was transfected to MC3T3-E1 cells with BMP2. The expression levels of miRNA-1-3p were determined by qRT-PCR. The expression of Runx2, OSX, OPN, and OCN was detected by Western blotting. ALP assay was performed to measure alkaline phosphatase activity. Calcium nodules were evaluated by alizarin red staining. Over-expression of hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) was performed and miRNA-1-3p rescue experiments were carried out. Over-expression of miRNA-1-3p promoted osteogenic differentiations and calcifications, as demonstrated by increased ALP, calcification and osteogenic markers. Knock-down of miRNA-1-3p generated the opposite results. HIF1AN was identified to be directly targeted by miRNA-1-3p. Over-expression of HIF1AN suppressed OD and calcifications, and miRNA-1-3p reversed the effect. Our results demonstrated that miRNA-1-3p could enhance OD of MC3T3-E1 cells through interacting with HIF1AN, which might be employed as therapeutic applications for bone formation and regeneration.

中文翻译：

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MicroRNA-1-3p 通过与缺氧诱导因子 1 α 抑制剂 (HIF1AN) 相互作用增强 MC3T3-E1 细胞的成骨细胞分化

研究证明miRNAs参与了成骨细胞分化（OD）的调控，miRNAs的异常表达与OD的各种状态有关。在这项研究中，我们使用 MC3T3-E1 细胞研究了 miRNA-1-3p 在 OD 中的作用。BMP2 用于诱导 MC3T3-E1 细胞的 OD。将 miRNA-1-3p 模拟物或 miRNA-1-3p 抑制剂转染到具有 BMP2 的 MC3T3-E1 细胞。通过qRT-PCR测定miRNA-1-3p的表达水平。通过Western印迹检测Runx2、OSX、OPN和OCN的表达。进行ALP测定以测量碱性磷酸酶活性。通过茜素红染色评估钙结节。进行了缺氧诱导因子 1-α 抑制剂 (HIF1AN) 的过表达，并进行了 miRNA-1-3p 拯救实验。miRNA-1-3p 的过表达促进了成骨分化和钙化，正如 ALP、钙化和成骨标志物增加所证明的那样。miRNA-1-3p 的敲除产生了相反的结果。HIF1AN 被鉴定为 miRNA-1-3p 直接靶向。HIF1AN 的过表达抑制了 OD 和钙化，而 miRNA-1-3p 则逆转了这种作用。我们的结果表明 miRNA-1-3p 可以通过与 HIF1AN 相互作用来增强 MC3T3-E1 细胞的 OD，这可能被用作骨形成和再生的治疗应用。和 miRNA-1-3p 逆转了这种作用。我们的结果表明，miRNA-1-3p 可以通过与 HIF1AN 相互作用来增强 MC3T3-E1 细胞的 OD，这可能被用作骨形成和再生的治疗应用。和 miRNA-1-3p 逆转了这种作用。我们的结果表明 miRNA-1-3p 可以通过与 HIF1AN 相互作用来增强 MC3T3-E1 细胞的 OD，这可能被用作骨形成和再生的治疗应用。