N6-methyladenosine-mediated Nrf2 Regulates the Defense Mechanism Against PM2.5-induced Pulmonary Fibrosis

Abstract Background: 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) modifications play 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 remain unknown. Here, we investigated the role and the underlying molecular mechanisms of m6A methylation of Nrf2 mRNA in PM2.5-induced PF. Results: Male C57BL/6 mice were exposed to filtered air (FA), unfiltered air (UA) and concentrated air (CA)for 16 weeks. 16HBE cells were treated with 0, 50, or 100 µg/mL PM2.5 for 24 h. Our data showed that chronic PM2.5 exposure could induce fibrosis in lung and increase Nrf2 signals. In Nrf2 deficient cells, α-SMA expression was significantly upregulated whereas E-cadherin decreased compared with WT cells after PM2.5 treatment which implied the aggravated fibrosis. m6A methyltransferase METTL3 was upregulated after PM2.5 treatment. m6A-methylated RNA immunoprecipitation (MeRIP) and qRT-PCR results showed that METTL3 improved the m6A modification of Nrf2 mRNA in PM2.5-exposed 16HBE cells. MeRIP-Seq and single-base T3 ligase-based PCR results showed that the m6A-modified sites of Nrf2 mRNA were 1317, 1376, and 935 in lung of mice after PM2.5 exposure. RIP results suggested that the m6A binding proteins YTHDF1/IGF2BP1 promoted Nrf2 translation by binding to Nrf2 mRNA m6A residues.Conclusions: Our results revealed the mechanism by which m6A regulated the activities of the Nrf2-mediated signaling pathway against PM2.5-induced PF.