The dynamic landscape of alternative splicing during silicosis progression.

BACKGROUND: Silicosis is a severe occupational pulmonary fibrosis disease caused by silica dust inhalation, characterized by persistent inflammation and progressive fibrosis. While transcriptomic studies have identified numerous genes dysregulated, molecular mechanisms extend beyond mere changes in gene expression levels. Recent evidence suggests that post-transcriptional regulatory mechanisms, particularly alternative splicing (AS), may play crucial roles in fibrotic processes. However, AS dynamics and functional significance in silicosis remain largely unexplored. METHOD: To investigate AS in silicosis pathogenesis, we analyzed RNA sequencing (RNA-Seq) data from a mouse model, delineating the dynamic landscape of gene expression and AS events across disease stages. AS events were classified into two categories: those correlated with and independent of gene expression changes. We integrated single-cell RNA sequencing (scRNA-seq) data to precisely map the cell type-specific expression patterns of these events. Through this analysis, we identified critical AS events, whose dynamic regulation and functional relevance during silicosis progression were validated using in vivo and in vitro models. RESULTS: A total of 26,232 high-confidence AS events were identified, with Skipping Exon (SE) being the predominant type (75.6%, 19,832/26,232). These splicing events were derived from 7,524 parental genes, with 72% (5,422) harboring at least two events, indicating widespread splicing complexity. 1,363 (5.2%) high-confidence events were significantly regulated across disease stages, with 88 Differentially Expressed Alternative Splicing (DEAS) events exhibiting a substantial splicing shift (absolute delta Percent Spliced In (PSI) greater than 0.2). Strikingly, only 11.4% (10/88) of these DEAS events showed concordant gene-level changes, while a substantial proportion (88.6%, 78/88) occurred despite stable gene level expression. Parental genes were enriched in silicosis-related pathways, including smooth muscle cell migration, actin bundle assembly and the small GTPase-mediated signal transduction. Integrated scRNA-seq helped map expression to specific cell types and prioritize validation targets. The dynamic regulation of three top targets, including Clec4e- Retained Intron (RI), Wdr37- Alternative First Exon (AFE), and Tns3-AFE, was validated in mouse models. In vitro functional experiments showed that Clec4e isoforms (RI and non-RI) possess distinct functional properties; dysregulation of Clec4e-RI may relieve inflammatory inhibition. These results suggest a potential regulatory role of AS reprogramming in silicosis pathogenesis and indicate Clec4e splicing events as a possible target for inflammation regulation. CONCLUSION: This study presents a landscape of AS in silicosis, demonstrating that AS constitutes a critical layer of pathological regulation distinct from transcriptional alterations. We identified and validated that the Clec4e-RI splicing isoform may play a key role in the silicosis pathogenesis by modulating macrophage-related inflammatory responses.