N6-methyladenosine (m6A) is a reversible modification commonly found in mammalian mRNAs, dynamically involved in many biological processes. The methyltransferase-like 16 (METTL16) is an m6A methyltransferase that has been proven to be closely associated with the occurrence of various diseases. We aimed to explore the role of METTL16 in airway inflammation of chronic obstructive pulmonary disease (COPD).

We screened out the relevant genes involved in m6A regulation from the Gene Expression Omnibus (GEO) database. The expression of METTL16 in COPD was verified using lung tissues of COPD patients and the mouse model of COPD induced by cigarette smoke (CS), as well as lung epithelial cell lines stimulated by cigarette smoke extract (CSE). The changes of pulmonary epithelial cell inflammation were analyzed upon METTL16 knockdown with lentiviral infection or METTL16 overexpression with plasmid transfection. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and metabolomic profiling were used to identify the potential regulatory pathways through METTL16. A 3-month COPD murine model was constructed to verify the antiinflammatory effects of glutamine-supplemented diet. Finally, RNA-seq and Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) were used to validate the potential targets of METTL16 for the glutamine metabolism pathway, and mitochondrial function was detected by the Seahorse Analyzer and JC-1 staining buffer.  

METTL16 was aberrantly decreased in lung tissues of COPD patients and the mouse model of COPD induced by cigarette smoke (CS). As similar, cigarette smoke extract (CSE) decreased METTL16 in lung epithelial cells. Knockdown of METTL16 aggravated CSE induced inflammation of pulmonary epithelial cell, while overexpression of METTL16 alleviated inflammation. MeRIP-seq suggested that METTL16 affected metabolic processes such as ATP synthesis, glycolysis, and amino acid transport. Metabolomic profiling pointed out the alanine, asparate and glutamate metabolism was the most significant metabolic pathway. Among them, the glutamine level was significantly reduced in the plasma of COPD patients, while the asparate level was significantly increased. Metabolic flux analysis indicated that METTL16 was involved in tricarboxylic acid (TCA) cycle. In addition, significant reduction in inflammation levels was observed in the 3-month COPD murine model fed a glutaminesupplemented diet. Furthermore, knockdown of METTL16 significantly reduced the stability of Glutamic-oxaloacetic transaminase 2 (GOT2) and downregulated its expression through m6A modification, while reprogramed glutamine metabolism in lung epithelial cells. Mechanistically, METTL16 regulated lung epithelial mitochondrial function by participating in the reprogramming of glutamine metabolism.  

Our study characterize the role of METTL16/GOT2/glutamine axis in the occurrence and development of COPD, and emphasize the potential value of METTL16 in the treatment of airway inflammations.