MicroRNA‐1225 activates Keap1‐Nrf2‐HO‐1 signalling to inhibit TNFα‐induced osteoclastogenesis by mediating ROS generation

Abstract

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.