State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
Department of Orthodontics, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, China.
Cell Biochem Funct. 2019 Jun;37(4):256-265. doi: 10.1002/cbf.3394. Epub 2019 Apr 24.
The influence of miRNA-1225-5p (miR-1225) and Keap1-Nrf2 signalling on in vitro osteoclast differentiation in mouse models was studied along with the underlying mechanism. In differentiated bone marrow-derived macrophages (BMMs), downregulated miR-1225 and upregulated Keap1 were observed in the course of receptor activator of nuclear factor kappa-Β ligand (RANKL)-mediated osteoclastogenesis. Bioinformatic analysis and dual-luciferase reporter assay showed that miR-1225 targeted the three prime untranslated region (3'-UTR) of Keap1 mRNA and caused its degradation. Transfection of a miR-1225 mimic or Keap1 silencing was found to inhibit osteoclastogenesis as evidenced by loss of activity and tartrate-resistant acid phosphatase (TRAP) staining, decreased expression of osteoclast markers, and associated genes and reduced number of multinuclear cells; in contrast, a miR-1225 inhibitor or Keap1 overexpression increased this process. In addition, transfection with the miR-1225 mimic or Keap1 silencing decreased the level of tumour necrosis factor (TNF)α, which was increased after miR-1225 inhibition and Keap1 overexpression. TNFα overexpression promoted Keap1 depletion-inhibited BMM osteoclastogenesis. Furthermore, reactive oxygen species (ROS) generation that is related to osteoclastogenesis and the Keap-Nrf2 axis was impaired by the miR-1225 mimic and Keap1 silencing, whereas it was increased following miR inhibition and overexpression of Keap1 and TNFα. Thus, miR-1225 inhibits osteoclastogenesis by directly activating the Keap1-Nrf2-HO-1 axis to repress TNFα-mediated ROS generation. SIGNIFICANCE OF THE STUDY: Our study showed that miR-1225 is significant in multiple malignancies and other pathological reactions. Transfection of a miR-1225 mimic or Keap1 silencing inhibits osteoclastogenesis. After miR-1225 inhibition and Keap1 overexpression, TNF was increased. TNFα overexpression promoted Keap1 depletion-inhibited BMM osteoclastogenesis. miR-1225 activates Keap1-Nrf2-HO-1 signal to inhibit TNFα-induced osteoclastogenesis.
研究了 microRNA-1225-5p（miR-1225）和 Keap1-Nrf2 信号对体外小鼠破骨细胞分化的影响及其潜在机制。在分化的骨髓来源巨噬细胞（BMM）中，在核因子 kappa-B 配体（RANKL）介导的破骨细胞发生过程中观察到 miR-1225 下调和 Keap1 上调。生物信息学分析和双荧光素酶报告基因分析表明，miR-1225 靶向 Keap1 mRNA 的 3'非翻译区（3'-UTR）并导致其降解。转染 miR-1225 模拟物或 Keap1 沉默被发现抑制破骨细胞生成，表现为活性丧失和抗酒石酸酸性磷酸酶（TRAP）染色减少，破骨细胞标志物和相关基因的表达减少，多核细胞数量减少；相反，miR-1225 抑制剂或 Keap1 过表达增加了这一过程。此外，转染 miR-1225 模拟物或 Keap1 沉默降低了肿瘤坏死因子（TNF）α的水平，而抑制 miR-1225 后和过表达 Keap1 后 TNFα的水平增加。TNFα过表达促进了 Keap1 耗竭抑制的 BMM 破骨细胞生成。此外，与破骨细胞发生和 Keap-Nrf2 轴相关的活性氧（ROS）生成被 miR-1225 模拟物和 Keap1 沉默所损害，而抑制 miR 后和过表达 Keap1 和 TNFα后 ROS 生成增加。因此，miR-1225 通过直接激活 Keap1-Nrf2-HO-1 轴来抑制 TNFα介导的 ROS 生成，从而抑制破骨细胞生成。研究意义：本研究表明，miR-1225 在多种恶性肿瘤和其他病理反应中具有重要意义。转染 miR-1225 模拟物或 Keap1 沉默抑制破骨细胞生成。抑制 miR-1225 后和过表达 Keap1 后，TNF 增加。TNFα过表达促进了 Keap1 耗竭抑制的 BMM 破骨细胞生成。miR-1225 激活 Keap1-Nrf2-HO-1 信号以抑制 TNFα诱导的破骨细胞生成。
