METTL3-m6A methylation inhibits Type II Alveolar Epithelial Cells proliferation and viability by promoting PTEN mRNA stability and translation efficiency in the acute lung injury

Abstract

Acute lung injury (ALI) is a life-threatening acute inflammatory disease with high morbidity and mortality rates. N6-methylation of adenosine (m6A) is an abundant mRNA nucleotide modification, which is critical for mRNA metabolism and function. However, the role of m6A modifications in ALI progression remains unclear. To address this problem, we modeled ALI in vivo by intraperitoneally injecting lipopolysaccharide (LPS) to C57BL/6 mice. In addition, we isolated alveolar type II cells (AECII) from the lungs of C57BL/6 mice and treated them in vitro with various concentrations of LPS. An m6A RNA methylation quantification kit was used to detect methylation levels. An m6A microarray and mRNA transcriptomic sequencing were used to screen the downstream targets of methyltransferase-like 3 (METTL3)-mediated m6A modifications. Gene and protein expression levels were determined by qRT-PCR and western blotting. The biological effects of modulating METTL3 expression level were evaluated both in vitro and in vivo. The binding between proteins and mRNA was verified by the RNA immunoprecipitation assay. The CCK-8 and EdU assays were used to examine changes in AECII viability and proliferation. The global number of mRNA m6A-modified loci and the “writer” methyltransferase METTL3 expression were significantly increased in the lung tissue following ALI and in LPS-treated AECII. METTL3 knockdown reduced the viability and proliferation of LPS-treated AECII. Furthermore, METTL3-mediated m6A modification of phosphatase and tensin homolog (Pten) mRNA enhanced its stability. Collectively, our results showed that METTL3 contributes to ALI progression by increasing stability and translation efficiency of Pten mRNA by means of m6A modification, indicating that METTL3 might become a new and innovative therapeutic target for the treatment of ALI.