Department of Orthopedics, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
Department of Gastroenterology, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
Mech Dev. 2020 Jun;162:103613. doi: 10.1016/j.mod.2020.103613. Epub 2020 May 6.
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.
研究证明，miRNAs 参与了成骨细胞分化（OD）的调节，miRNAs 的异常表达与 OD 的各种状态有关。在本研究中，我们使用 MC3T3-E1 细胞研究了 miRNA-1-3p 在 OD 中的作用。使用 BMP2 诱导 MC3T3-E1 细胞的 OD。用 BMP2 将 miRNA-1-3p 模拟物或 miRNA-1-3p 抑制剂转染至 MC3T3-E1 细胞。通过 qRT-PCR 测定 miRNA-1-3p 的表达水平。通过 Western blot 检测 Runx2、OSX、OPN 和 OCN 的表达。通过碱性磷酸酶（ALP）测定法测定碱性磷酸酶活性。通过茜素红染色评估钙结节。进行低氧诱导因子 1 抑制剂（HIF1AN）过表达和 miRNA-1-3p 挽救实验。过表达 miRNA-1-3p 可促进成骨分化和钙化，ALP、钙化和成骨标志物增加表明这一点。miRNA-1-3p 的敲低则产生相反的结果。鉴定出 HIF1AN 是 miRNA-1-3p 的直接靶标。HIF1AN 的过表达抑制 OD 和钙化，而 miRNA-1-3p 则逆转了这一作用。我们的研究结果表明，miRNA-1-3p 可通过与 HIF1AN 相互作用增强 MC3T3-E1 细胞的 OD，这可能被用于骨形成和再生的治疗应用。
