Phlda1 stabilized by Mettl14-mediated m6A modification promotes lipopolysaccharide-induced acute lung injury

Pleckstrin homology-like domain family A member 1 (Phlda1) has been reported to be involved in regulating cell proliferation, death, immune responses, and oxidative stress. Here, this study investigated the functions and mechanisms of Phlda1 in acute lung injury (ALI). Lipopolysaccharide (LPS)-induced murine alveolar epithelial MLE-12 cells and NLRP3 inflammasome activation model using the mouse macrophage cell line RAW264.7 in vitro were established. Levels of Phlda1 and Mettl14 were examined using qRT-PCR and western blotting. Cell viability and apoptosis were determined by CCK-8 assay and flow cytometry. ELISA analysis was performed to measure the expression of inflammatory factors. Oxidative stress was evaluated by determining the production of reactive oxygen species and malondialdehyde. The N6-methyladenosine (m6A) modification profile was determined by methylated RNA immunoprecipitation assay. The interaction between Mettl14 and Phlda1 was validated by the dual-luciferase reporter assay. Levels of Phlda1 were higher in LPS-induced lung epithelial cells and the NLRP3 inflammasome activation model. Functionally, silencing of Phlda1 reversed LPS-induced apoptosis, inflammation, and oxidative stress in epithelial cells, and suppressed LPS-mediated activation of the NLRP3 inflammasome in RAW264.7. Mechanistically, Mettl14 stabilized Phlda1 expression by inducing Phlda1 m6A modification. Mettl14 knockdown suppressed LPS-induced lung epithelial cell injury and NLRP3 inflammasome activation, while these effects were abolished by Phlda1 overexpression. Collectively, Phlda1 was stabilized by Mettl14-mediated m6A modification and then promoted LPS-induced lung epithelial cell injury and NLRP3 inflammasome activation, recommending a novel insight into the development of therapeutic strategies for the prevention of LPS-induced ALI.