N⁶-methyladenosine mediates Nrf2 protein expression involved in PM2.5-induced pulmonary fibrosis

It has been reported that particulate matter with an aerodynamic diameter of <2.5 µm (PM2.5) could induce epithelial-mesenchymal transition (EMT)- and extracellular matrix (ECM)-related pulmonary fibrosis (PF). The transcription factor Nrf2 alleviated PM2.5-induced PF by antagonizing oxidative stress. The N⁶-methyladenosine (m⁶A) modification plays a significant role in the stress response. However, the effect of m⁶A modification on the mechanisms of Nrf2-mediated defense against PM2.5-induced PF remained unknown. Here, we explored the role and the underlying molecular mechanisms of m⁶A methylation of Nrf2 mRNA in PM2.5-induced PF. We established filtered air (FA), unfiltered air (UA), and concentrated PM2.5 air (CA) group mice model and 0, 50, and 100 μg/mL PM2.5-treated 16HBE cell models. The extent of lung fibrosis in mice and fibrosis indicators were detected by histopathological analysis, immunohistochemical staining and western blotting. The molecular mechanism of m⁶A-modified Nrf2 was demonstrated by m⁶A-methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), qRT-PCR and T3 ligase-based PCR. Our data showed that PM2.5 exposure for 16 weeks could induce pulmonary fibrosis and activate Nrf2 signaling pathway. m⁶A methyltransferase METTL3 was upregulated after PM2.5 treatment in vivo and in vitro. Moreover, METTL3 mediated m⁶A modification of Nrf2 mRNA and promoted Nrf2 translation in mice and 16HBE cells after PM2.5 exposure. Mechanistically, three m⁶A-modified sites (1317, 1376 and 935; numbered relative to the first nucleotide of 3'UTR) of Nrf2 mRNA were identified in PM2.5-treatment 16HBE cells. Furthermore, the m⁶A binding proteins YTHDF1/IGF2BP1 promoted Nrf2 translation by binding to m⁶A residues of Nrf2 mRNA. Our results revealed the mechanism of m⁶A mediated Nrf2 signaling pathway against oxidative stress, which affected the development of PM2.5-induced PF.