Insights into the mechanism underlying crystalline silica-induced pulmonary fibrosis via transcriptome-wide m6A methylation profile

Abstract

Silicosis is one of the most severe interstitial lung fibrosis diseases worldwide, caused by crystalline silica exposure. While the mechanisms and pathogenesis underlying silicosis remained unknown. N⁶-methyladenosine (m⁶A) methylation has received significant attention in a variety of human diseases. However, whether m⁶A methylation is involved in silicosis has not been clarified. In this study, we conducted methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and transcriptome sequencing (RNA-Seq) to profile the m⁶A modification in normal and silicosis mouse models (n = 3 pairs). The global levels of m⁶A methylation were further assessed by m⁶A RNA methylation quantification kits, and the major regulators of m⁶A RNA methylation were verified by qRT-PCR. Our results showed that long-term exposure to crystalline silica led to silicosis, accompanied by increasing levels of m⁶A methylation. Upregulation of METTL3 and downregulation of ALKBH5, FTO, YTHDF1, and YTHDF3 might contribute to aberrant m⁶A modification. Compared with controls, 359 genes showed differential m⁶A methylation peaks in silicosis (P < 0.05 and FC ≥ 2). Among them, 307 genes were hypermethylated, and 52 genes were hypomethylated. RNA-Seq analysis revealed 1091 differentially expressed genes between the two groups, 789 genes were upregulated and 302 genes were downregulated in the lungs of silicosis mice (P < 0.05 and FC ≥ 2). In the conjoint analysis of MeRIP-Seq and RNA-Seq, we identified that 18 genes showed significant changes in both m⁶A modification and mRNA expression. The functional analysis further noted that these 18 m⁶A-mediated mRNAs regulated pathways that were closely related to "phagosome", "antigen processing and presentation", and "apoptosis". All findings suggested that m⁶A methylation played an essential role in the formation of silicosis. Our discovery with multi-omics approaches not only gives clues for the epigenetic mechanisms underlying the pathogenesis of silicosis but also provides novel and viable strategies for the prevention and treatment of silicosis.

Keywords: Crystalline silica; M(6)A RNA methylation; Pulmonary fibrosis; Silicosis.