Identification of hub genes and lncRNAs driving the transition from early response to methotrexate resistance in HT29 colon cancer cells

Resistance to methotrexate (MTX) hampers therapeutic efficacy in colorectal cancer. The molecular mechanisms underlying adaptive drug resistance remain poorly characterized. In this study, we aimed to identify genes associated with early transcriptional response, to uncover lncRNA-mRNA regulatory networks linked to MTX resistance, and to validate their functional contribution using Polypurine reverse Hoogsteen (PPRH) oligonucleotides as gene-silencing tools. Parental HT29 cells were incubated with MTX for short periods of time (8, 24, 48 h), and transcriptomic changes were assessed by whole genome microarrays. Differentially expressed genes (DEGs) were analyzed for functional enrichment (GSEA and STRING). Additionally, long non-coding RNA (lncRNA) expression was evaluated using qPCR SignArrays at 24 and 48 h after MTX treatment both in HT29 parental and resistant cells. Differentially expressed lncRNAs in both conditions were associated with protein-coding genes (ENCORI StarBase) which were then intersected with the DEGs found in MTX-responsive HT29 cells. PPRHs were designed against selected targets, and their effects on gene expression and cell viability were assessed by RT-qPCR and cytotoxicity assays. MTX induced transcriptional remodeling involving stress-response pathways, regulation of signaling, and cell communication including proliferation and migration. Caveolin-1 (CAV1) emerged as a protein-coding hub present in both early adaptive and resistant cells. MALAT1 and NEAT1 were overexpressed and identified as lncRNA hubs, whereas H19 displayed a dual expression pattern, overexpressed in sensitive cells 48 h post-MTX exposure but underexpressed in resistant cells. Our findings support the involvement of these lncRNAs and CAV1 in the early transcriptional response to MTX in colorectal cancer cells.