Outline of Transcripts and Genes in HBECs
A total of 32,113 transcripts and 18,809 genes were screened out from the HBECs in both SMV-treated and control groups (Supplementary file 1 and 2). Fig. 1A-F indicated the UMAP, expression density, probes distribution, mean-variance trend, moderated T statistic, and the volcano plot of the DEGs, respectively.
Totally 513 differentially expressed transcripts (Supplementary file 3; 232 down-regulated and 281 up-regulated) and 455 DEGs (Supplementary file 4; 201 down-regulated and 254 up-regulated) were identified. The top 25 up- and down-regulated genes in SMV-treated HBECs were shown in Tables 2 and 3, respectively.
GO Enrichment for DEGs
As indicated in Fig. 2A and Supplementary file 5, upregulated genes might be related to cell adhesion, RNA processing, angiogenesis, response to xenobiotic stimulus, the regulation of cell proliferation, and cell proliferation. Also, down-regulated genes were likely to involve signal transduction, response to drug, cell-cell adhesion, cell adhesion, transcription regulation, NF-κB transcription factor activity, and mitochondrion localization (Fig. 2B and Supplementary file 6). In addition, the expression profiles of the DEGs in cell adhesion, cell migration, the positive regulation of cell migration, the negative regulation of cell proliferation, the regulation of cell proliferation, and the positive regulation of cell proliferation were visualized in Fig. 2C-G with heatmaps, respectively.
KEGG Enrichment for DEGs
Upregulated genes in SMV-treated HBECs mainly participated in metabolic pathway, MAPK, Wnt, Rap1, ErbB, FoxO, cAMP, Ras, PI3K-Akt, relaxin, apelin, calcium, phagosome, axon guidance, focal adhesion, non-small cell lung cancer (NSCLC), transcriptional misregulation in cancer, regulation of actin cytoskeleton, drug metabolism-cytochrome P450, complement and coagulation cascades, small cell lung cancer (SCLC), pancreatic secretion, thyroid hormone, vascular smooth muscle contraction, glycerolipid metabolism, and cortisol synthesis and secretion signaling pathways (Fig. 3A and Supplementary file 7).
Also, as shown in Fig. 3B and Supplementary file 8, the downregulated genes in SMV-treated HBECs mainly related to oxytocin, Wnt, relaxin, calcium, PPAR, thyroid hormone, metabolic pathways, NOD-like receptor, axon guidance, NF-κB, CAMs, transcriptional misregulation in cancer, glutathione metabolism, focal adhesion, mucin type O-glycan biosynthesis, cholinergic synapse, glutamatergic synapse, platelet activation, fluid shear stress and atherosclerosis, arginine biosynthesis, lysine degradation, basal cell carcinoma, acute myeloid leukemia, and long-term potentiation.
Moreover, Fig. 3C-I represented the heatmaps of the DEGs in MAPK, metabolic pathways, NSCLC, PI3K-Ak, SCLC, CAMs, and PPAR signaling pathways, respectively.
Reactome enrichment and Protein classification for DEGs
The upregulated DEGs had a vital role in many signaling pathways, including metabolism, GPCR downstream signaling, developmental biology, signaling by receptor tyrosine kinases, extracellular matrix organization, disease, Axon guidance, hemostasis, signaling by GPCR, metabolism of lipids, diseases of signal transduction, GPCR ligand binding, L1CAM interactions, neuronal system, ECM proteoglycans, platelet activation, signaling and aggregation (Fig. 4A).
The down-regulated DEGs played an important role in gene expression, immune system, signal transduction, metabolism, metabolism of proteins, innate immune system, developmental biology, neuronal system, neutrophil degranulation, adaptive immune system, hemostasis, interferon signaling, transmission across chemical synapses, extracellular matrix organization, G alpha (i) signalling events, inositol phosphate metabolism, G-protein mediated events, peptide hormone metabolism, opioid signaling, and stimuli-sensing channels (Fig. 4B).
The upregulated DEGs in SMV-treated HBECs were mainly involved in integrin, metalloprotease, intercellular signal molecule, g-protein coupled receptor, extracellular matrix protein, cell adhesion molecule, protease, growth factor, membrane-bound signaling molecule, histone modifying enzyme, lambda repressor-like transcription factor, and oxidase (Fig. 4C).
Down-regulated DEGs in SMV-treated HBECs played a significant role in cell adhesion molecule, cadherin, DNA-binding transcription factor, C2H2 zinc finger transcription factor, gene-specific transcriptional regulator, glycosyltransferase, Zinc finger transcription factor, immunoglobulin superfamily cell adhesion molecule, actin binding motor protein, general transcription factor, ion channel, and membrane-bound signaling molecule (Fig. 4D).
Integration of the PPI Network
To further discover the key genes linked to SMV-induced pulmonary toxicity, PPI networks of the DEGs were constructed. as shown in Fig. 5A and B, a set of upregulated genes (including LAMC2, ITGA5, LAMB1, LOX, ITGB3, ITGA2, HSPG2, COL5A2, MMP2, and LAMB3, et al) and downregulated genes (including GLUL, BIRC3, IL7R, BRCA1, PAX6, WT1, GPX7, and KAT2A, et al) might play a vital role in SMV-induced pulmonary toxicity.
Hub genes and their functions
The hub genes associated with SMV-induced pulmonary toxicity were obtained based on the MCC method. As shown in Fig. 6A and Table 4, the top 20 hub genes upregulated in SMV-treated HBECs included ITGA5, ITGB3, ITGA2, LAMB1, LAMC2, HSPG2, COL5A2, MMP2, LOX, LAMB3, SNAI2, ACTA2, LOXL2, ITGBL1, TGFBR2, LOXL1, JAG1, WNT5A, LTBP1, and EREG. In addition, the top 20 hub genes downregulated in SMV-treated HBECs included ICAM1, ITGA4, ASS1, COL1A1, NCAM1, IL7R, BRCA1, PAX6, GLUL, BIRC3, WT1, GPX7, KAT2A, NPPB, ALDH2, NRXN3, E2F5, SYDE2, SULF1, and ACTC1 (Fig. 6B and Table 5).
GO enrichment suggested that the top 20 upregulated hub genes played a key role in animal organ morphogenesis, endodermal cell differentiation, peptidyl-lysine oxidation, collagen fibril organization, angiogenesis, cell migration, cell-substrate adhesion, substrate adhesion-dependent cell spreading, keratinocyte differentiation, and the response to xenobiotic stimulus, et al (Fig. 6C).
Similarly, the top 20 downregulated hub genes participated in the positive regulation of gene expression, response to drug, regulation of cell cycle, response to ionizing radiation, kidney development, negative regulation of centriole replication, diaphragm development, glomerular basement membrane development, cell adhesion, leukocyte migration, positive regulation of vascular endothelial growth factor production, leukocyte cell-cell adhesion, embryonic skeletal system development, and blood vessel development (Fig. 6D).
The top 20 upregulated hub genes participated in various signing pathways, such as PI3K-Akt, TGF-beta, Hippo, apelin, MAPK, toxoplasmosis, focal adhesion, endocrine resistance, platelet activation, SCLC, ECM-receptor interaction, relaxin, phagosome, regulation of actin cytoskeleton, osteoclast differentiation signaling pathways (Fig. 8E).
Meanwhile, the top 20 down-regulated Hub genes had a major role in PI3K-Akt, NF-κB, TNF, metabolic pathways, focal adhesion, arginine biosynthesis, alanine, aspartate and glutamate metabolism, platinum drug resistance, biosynthesis of amino acids, ECM-receptor interaction, fluid shear stress and atherosclerosis, and necroptosis signaling pathways (Fig. 8F).