RNA-Sequence Analysis of Caco-PXR Cell Line Exposed to Okadaic Acid

Okadaic acid (OA), a polyether marine toxin responsible for diarrhetic shellfish poisoning (DSP), poses a significant public health concern. While its inhibitory action on serine/threonine phosphatases, particularly PP2A and PP1 is well established, its genome-wide transcriptional impact on intestinal xenobiotic sensing pathways particularly via the human pregnane-X receptor (PXR) remains insufficiently characterized. This study therefore employed high throughput RNA sequencing (RNA-seq) to evaluate transcriptional responses in the Caco-2 cell line stably transfected to overexpress PXR (Caco-PXR) following 24-hour exposure to OA of varying concentration gradients (0 nM, 20 nM, 80 nM, 160 nM and 600 nM). Differential gene expression and Gene Ontology (GO) pathway analysis was performed to understand the modulation of genes, the biological processes and pathways they significantly alter upon exposure. Principal component analysis showed clear dose-dependent clustering of samples, indicating that OA exposure was the dominant driver of transcriptional variation. Results from the differential gene expression analysis showed an observable dose-dependent transcriptional modulation of the differentially expressed genes (DEGs). OA exposure at 20 nM resulted in the least number of DEGs (8), 487 DEGs were modulated at 80 nM, 3,037 DEGs at 160 nM and 600 nM OA exposure resulted in the highest number of DEGS (6,079). At 20 nM, all DEGs were upregulated, including genes associated with ERK/MAPK feedback and early stress signalling like DUSP6 and FOSL1. At 80nM, the response remained predominantly upregulated, whereas 160 nM and 600 nM exposure yielded increasingly balanced then predominantly repressive transcriptional responses. The functional enrichment analysis indicated that lower OA concentration mainly activated signalling, locomotion, angiogenesis, epithelial remodeling, and ERK1/2 cascade-related processes. In contrast, higher OA concentration, especially 600 nM, were associated with a broader disruption of cell-cycle regulation, DNA metabolic process, inflammatory and immune-related pathways, epithelial-to-mesenchymal transition-related processes and suppression of fatty acid, steroid, xenobiotic and small-molecule metabolic pathways. These findings suggest that OA induces a progressive transcriptional shift within the Caco-PXR cell line, moving from protein phosphatase (PP1/PP2A) inhibition, hyperphosphorylation and adaptive kinase-linked signalling towards widespread inflammatory, metabolic and epithelial dysfunction at higher concentrations. Overall, this study demonstrates that RNA-seq can reveal dose-dependent molecular signatures of OA induced toxicity within the Caco-PXR cell line.