Differential gene expression and enrichment analysis
No genes were identified that were differentially expressed with a false discovery rate (FDR) less than 0.05 following PDT in the pooled analysis of the fibroblast and cSCC cell lines (Supplemental Table 2). The FDR is an adjusted p-value after correcting for multiple testing. As a result, transcription factor, gene enrichment, and pathway analysis were performed with genes with a p-value less than 0.05. Eighty-three genes were identified to be upregulated and 134 were found to be downregulated (Figure 1D).
Pathways related to cell division and proliferation were found to be downregulated. The specific pathways that were significantly downregulated with an FDR value <0.05 in KEGG 2021 included “Cell Cycle” (Figure 2A). GO Biological Processes 2021 pathways that were downregulated include microtubule cytoskeleton organization involved in mitosis (GO:1902850), mitotic spindle organization (GO:0007052), and mitotic nuclear division (GO:0140014) (Figures 2C). Supplemental Table 3 includes the full list of downregulated GO Biological Processes 2021. No KEGG 2021 or GO Biological Processes 2021 were significantly enriched (FDR < 0.05) in the upregulated genes (Figures 2B and 2D). KEGG 2021 pathways with a p <0.05 include mitophagy (FDR=0.21) and autophagy (FDR=0.33).
Transcription factors were analyzed using consensus genes targets in the ENCODE and ChEA 2022 databases. Following ALA PDT, 23 transcription factors were significantly enriched in downregulated genes (Supplemental Table 4). The transcription factors with the most significant enrichment included E2F4, FOXM1, Sin3a, and NFYA/B (Figure 3A). Figure 3C demonstrates the clustergram of genes commonly downregulated among the enriched transcription factors in the ENCODE and ChEA databases. Upregulated transcription factors included CPEBP (FDR=0.60) and NFE2L2 (FDR=0.29) but were not significant (Figures 3C and D).