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

Cell Biochem Funct. 2019 Jun;37(4):256-265. doi: 10.1002/cbf.3394. Epub 2019 Apr 24.

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.

Keywords: Keap1-Nrf2-HO-1; ROS; TNFα; bone marrow-derived macrophages; miR-1225; osteoclastogenesis.

MeSH terms

  • Animals
  • Cells, Cultured
  • Heme Oxygenase-1 / metabolism*
  • Humans
  • Kelch-Like ECH-Associated Protein 1 / metabolism*
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Inbred BALB C
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • NF-E2-Related Factor 2 / metabolism*
  • Osteogenesis*
  • Reactive Oxygen Species / metabolism*
  • Receptor Activator of Nuclear Factor-kappa B / metabolism
  • Signal Transduction*
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism*

Substances

  • Keap1 protein, mouse
  • Kelch-Like ECH-Associated Protein 1
  • MIRN1225 microRNA, human
  • Membrane Proteins
  • MicroRNAs
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • Reactive Oxygen Species
  • Receptor Activator of Nuclear Factor-kappa B
  • Tumor Necrosis Factor-alpha
  • Heme Oxygenase-1
  • Hmox1 protein, mouse