Transcriptomic analysis of lung fibroblasts transduced with a N4ICD
The identification of differentially expressed genes (DEGs) was conducted by comparing the RNA sequencing data from samples with activated N4ICD and control samples pCIG. DEGs were selected based on the following threshold values: |logFC| > 1.5 and adjusted p-value < 0.05. According to PCA and sPLS-DA, the samples formed two clusters - pCIG and N4ICD (Fig. 3a, b).
In the samples with activated N4ICD, 640 DEGs were identified, with 413 exhibiting positive regulation and 227 showing negative regulation, indicative of significant changes in the transcriptional landscape. Among the DEGs, genes serving as targets of Notch signaling, including HEYL (logFC = 9.13, p.adj = 1.28e-11), HES1 (logFC = 6.39, p.adj = 2.43e-45), HES4 (logFC = 4.44, p.adj = 4.61e-31), and the upregulation of the NOTCH3 gene (logFC = 5.07, p.adj = 1.39e-148) (Fig. 3c, d) were present, confirming the activation of Notch signaling in N4ICD samples.
Significantly, in N4ICD-induced samples, there is an upregulation in the expression of genes associated with fibrosis (ELN, logFC = 8.62, p.adj = 2.83e-158; COL1A1, logFC = 4.04, p.adj = 7.10e-45; ACTA2, logFC = 3.96 p.adj = 1.06e-36) and collagen synthesis (Table 1, Fig. 3c, d). Additionally, several genes are identified as markers of myofibroblasts (WNT5A, logFC = 2.36, p.adj = 8.40e-11; ACTG2, logFC = 4.31, p.adj = 8.86e-26; CNN1, logFC = 5.78, p.adj = 2.14e-63), particularly those marking early myofibroblasts (ACTA2, logFC =3.96, p.adj = 1.06e-36; TAGLN, logFC = 1.91, p.adj = 2.47e-9).18 Alongside fibrotic markers, genes related to TGFβ signaling, which mediate cell differentiation into myofibroblasts, were identified (TGFβ3, logFC = 2.70, p.adj = 3.14e-9; TGFβ2, logFC = 1.44, p.adj = 0.0001; TGFβ1, logFC = 1.37, p.adj = 1.73e-9; SKIL, logFC = 1.87, p.adj = 1.88e-5; INHBA, logFC = 3.87, p.adj = 0.0008; SERPINE1, logFC = 1.59, p.adj = 1.66e-9). Lastly, the RhoA pathway was examined, specifically noting the expression level of the SFRP1 gene (logFC = −2.39, p.adj = 4.99e-20), known to suppress myofibroblast invasiveness.19
Table 1Genes encoding collagens activated in N4ICD-transduced cells
Collagens | N4ICD vs pCIG (logFC) |
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COL5A3 | 8.03 |
COL14A1 | 5.29 |
COL3A1 | 4.29 |
COL1A1 | 4.04 |
COL5A1 | 3.41 |
COL3A1_1 | 3.27 |
COL4A2 | 3.24 |
COL18A1_1 | 2.54 |
COL5A2 | 2.54 |
COL1A2 | 2.33 |
COL4A6 | −2.21 |
COL6A3 | 1.72 |
COL6A1 | 1.67 |
Enrichment analysis of the biological processes ontology reveals that positively regulated DEGs are involved in regulating Notch signaling, ECM metabolism, and collagen fibril assembly (Fig. 4a). The correlation of DEGs with relevant pathways according to the Reactome Pathway database aligns with the ontology-based analysis, revealing enrichment in pathways such as ECM organization, collagen biosynthesis and formation, and assembly of collagen fibrils (Fig. 4b).
Based on the identified DEGs, it can be inferred that the investigated cells exhibit the phenotype of highly invasive myofibroblasts. Typically, these cells appear during trauma to regenerate damaged tissues. However, in fibrosis, they accumulate, leading to increased production of ECM.