Department of Orthopedic, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Department of Nephrology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Endocrinol Metab (Seoul). 2023 Feb;38(1):156-173. doi: 10.3803/EnM.2022.1516. Epub 2023 Jan 6.
Evidence has revealed the involvement of microRNAs (miRNAs) in modulating osteogenic differentiation, implying the promise of miRNA-based therapies for treating osteoporosis. This study investigated whether miR-181a-5p influences osteogenic differentiation and bone formation and aimed to establish the mechanisms in depth.
Clinical serum samples were obtained from osteoporosis patients, and MC3T3-E1 cells were treated with osteogenic induction medium (OIM) to induce osteogenic differentiation. miR-181a-5p-, Runt-related transcription factor 1 (Runx1)-, and/or allograft inflammatory factor-1 (AIF-1)-associated oligonucleotides or vectors were transfected into MC3T3-E1 cells to explore their function in relation to the number of calcified nodules, alkaline phosphatase (ALP) staining and activity, expression levels of osteogenesis-related proteins, and apoptosis. Luciferase activity, RNA immunoprecipitation, and chromatin immunoprecipitation assays were employed to validate the binding relationship between miR-181a-5p and Runx1, and the transcriptional regulatory relationship between Runx1 and AIF-1. Ovariectomy (OVX)-induced mice were injected with a miR-181a-5p antagonist for in vivo verification.
miR-181a-5p was highly expressed in the serum of osteoporosis patients. OIM treatment decreased miR-181a-5p and AIF-1 expression, but promoted Runx1 expression in MC3T-E1 cells. Meanwhile, upregulated miR-181a-5p suppressed OIM-induced increases in calcified nodules, ALP content, and osteogenesis-related protein expression. Mechanically, miR-181a-5p targeted Runx1, which acted as a transcription factor to negatively modulate AIF-1 expression. Downregulated Runx1 suppressed the miR-181a-5p inhibitor-mediated promotion of osteogenic differentiation, and downregulated AIF-1 reversed the miR-181a-5p mimic-induced inhibition of osteogenic differentiation. Tail vein injection of a miR-181a-5p antagonist induced bone formation in OVX-induced osteoporotic mice.
In conclusion, miR-181a-5p affects osteogenic differentiation and bone formation partially via the modulation of the Runx1/AIF-1 axis.
有证据表明 microRNAs（miRNAs）参与调节成骨分化，这意味着 miRNA 疗法有望用于治疗骨质疏松症。本研究旨在探讨 miR-181a-5p 是否影响成骨分化和骨形成，并深入研究其机制。
从骨质疏松症患者中获取临床血清样本，并使用成骨诱导培养基（OIM）处理 MC3T3-E1 细胞以诱导成骨分化。将 miR-181a-5p、Runt 相关转录因子 1（Runx1）和/或同种异体炎症因子 1（AIF-1）相关的寡核苷酸或载体转染到 MC3T3-E1 细胞中，以探讨它们与钙化结节数量、碱性磷酸酶（ALP）染色和活性、成骨相关蛋白表达和细胞凋亡的关系。利用荧光素酶活性、RNA 免疫沉淀和染色质免疫沉淀实验验证 miR-181a-5p 与 Runx1 的结合关系，以及 Runx1 与 AIF-1 的转录调控关系。通过尾静脉注射 miR-181a-5p 拮抗剂对 OVX 诱导的骨质疏松症小鼠进行体内验证。
骨质疏松症患者血清中 miR-181a-5p 表达水平升高。OIM 处理降低了 MC3T-E1 细胞中 miR-181a-5p 和 AIF-1 的表达，但促进了 Runx1 的表达。同时，上调 miR-181a-5p 抑制了 OIM 诱导的钙化结节、ALP 含量和成骨相关蛋白表达的增加。机制上，miR-181a-5p 靶向 Runx1，作为转录因子负调控 AIF-1 的表达。下调 Runx1 抑制了 miR-181a-5p 抑制剂促进的成骨分化，下调 AIF-1 逆转了 miR-181a-5p 模拟物诱导的成骨分化抑制。尾静脉注射 miR-181a-5p 拮抗剂可诱导 OVX 诱导的骨质疏松症小鼠的骨形成。
综上所述，miR-181a-5p 通过调节 Runx1/AIF-1 轴影响成骨分化和骨形成。
