Department of Orthopedics, The First Affiliated Hospital of Henan University, Kaifeng, Henan 475001, P.R. China.
Department of Pharmacy, The First Affiliated Hospital of Henan University, Kaifeng, Henan 475001, P.R. China.
Mol Med Rep. 2020 Mar;21(3):1125-1132. doi: 10.3892/mmr.2020.10938. Epub 2020 Jan 13.
The present study aimed to investigate the effects of microRNA (miR)‑21 on osteoclastogenesis and its underlying molecular mechanisms. The expression levels of tartrate‑resistant acid phosphatase (TRAP) and miR‑21 were detected during osteoclastogenesis in receptor activator of NF‑κB ligand (RANKL)‑induced RAW264.7 cells via reverse transcription‑quantitative PCR. Bioinformatics and dual luciferase reporter assays were performed to analyze the association between miR‑21 and Pten. RANKL‑induced RAW264.7 cells were divided into the following groups: MiR‑negative control (NC), miR‑21 mimic, miR‑21 inhibitor and miR‑21 mimic + LY294002. The effects of miR‑21 on osteoclastogenesis and bone resorption were then detected using TRAP staining and a bone resorption assay. Pten, phosphorylated‑Akt and nuclear factor of activated T cell (NFATc1) expression levels were measured by western blotting to analyze the effects of miR‑21 on the PI3K/Akt signaling pathway. The present study revealed that miR‑21 was upregulated during osteoclastogenesis in RANKL‑induced RAW264.7 cells. Furthermore, miR‑21 negatively regulated Pten. Compared with the miR‑negative control (NC) group, the number of osteoclasts and the percentage of bone resorption were increased in the miR‑21 mimic group, whereas they were decreased in the miR‑21 inhibitor group. The number of osteoclasts and the percentage of bone resorption in the miR‑21 mimic + LY294002 group were lower than in the miR‑21 mimic group. Compared with the miR‑NC group, the protein expression levels of Pten were decreased, whereas p‑Akt and NFATc1 were increased in the miR‑21 mimic group. Conversely, Pten protein expression was increased, whereas p‑Akt and NFATc1 were decreased in the miR‑21 inhibitor group. In the miR‑21 mimic + LY294002 group, Pten protein expression was higher, and p‑Akt and NFATc1 were lower than in the miR‑21 mimic group. In conclusion, miR‑21 is upregulated during osteoclastogenesis, and may promote osteoclastogenesis and bone resorption through activating the PI3K/Akt signaling pathway via targeting Pten.
本研究旨在探讨微小 RNA（miR）-21 对破骨细胞生成的影响及其潜在的分子机制。通过逆转录定量聚合酶链反应检测核因子κB 受体激活物配体（RANKL）诱导的 RAW264.7 细胞破骨细胞生成过程中抗酒石酸酸性磷酸酶（TRAP）和 miR-21 的表达水平。通过生物信息学和双荧光素酶报告基因检测分析 miR-21 与 Pten 之间的关联。将 RANKL 诱导的 RAW264.7 细胞分为以下几组：miR-阴性对照（NC）组、miR-21 模拟物组、miR-21 抑制剂组和 miR-21 模拟物+LY294002 组。然后通过 TRAP 染色和骨吸收测定检测 miR-21 对破骨细胞生成和骨吸收的影响。通过 Western blot 检测 Pten、磷酸化 Akt 和激活 T 细胞核因子（NFATc1）的表达水平，分析 miR-21 对 PI3K/Akt 信号通路的影响。本研究表明，在 RANKL 诱导的 RAW264.7 细胞破骨细胞生成过程中，miR-21 上调。此外，miR-21 负调控 Pten。与 miR-NC 组相比，miR-21 模拟物组破骨细胞数量和骨吸收百分比增加，而 miR-21 抑制剂组减少。miR-21 模拟物+LY294002 组的破骨细胞数量和骨吸收百分比低于 miR-21 模拟物组。与 miR-NC 组相比，miR-21 模拟物组 Pten 蛋白表达降低，而磷酸化 Akt 和 NFATc1 表达增加。相反，miR-21 抑制剂组 Pten 蛋白表达增加，而磷酸化 Akt 和 NFATc1 表达减少。在 miR-21 模拟物+LY294002 组中，Pten 蛋白表达高于 miR-21 模拟物组，磷酸化 Akt 和 NFATc1 表达低于 miR-21 模拟物组。综上所述，miR-21 在破骨细胞生成过程中上调，可能通过靶向 Pten 激活 PI3K/Akt 信号通路促进破骨细胞生成和骨吸收。
