Time-Resolved Transcriptomics Reveal Spliceosomal Disruption and Senescence Pathways in Crocin-Treated Hepatocellular Carcinoma Cells

Abstract

Saffron-derived crocin exhibits anti-cancer properties, but the pathways underlying its effects remain incompletely characterized. Here, we utilized a high-dose perturbation strategy (1-2 mM crocin) to probe maximal pathway engagement in HepG2 hepatocellular carcinoma cells via time-series transcriptomics. We treated cells for 2, 6, 12, and 24 h and analyzed transcriptomic and splicing profiles at each timepoint. We identified 7400-12,100 differentially expressed genes (DEGs) per condition, with the higher dose (CR2) producing more total DEGs but the lower dose (CR1) demonstrating differential pathway prioritization. The spliceosome pathway ranked first among downregulated pathways for CR1 across multiple timepoints (false discovery rate, FDR p = 10-21 to 10-36) but only fourth for CR2, suggesting dose-dependent differences in pathway prioritization. Differential splicing analysis revealed functional spliceosome disruption, with 2000-2600 significant exon skipping events per condition and aberrant splicing of spliceosome components including HNRNPH1 (change in percent spliced in, dPSI = -0.78 to -0.89). Additionally, 66 genes implicated in non-alcoholic fatty liver disease were downregulated at 24 h (FDR p = 8x10-8). Crocin exposure consistently downregulated spliceosomal machinery genes while upregulating senescence and autophagy pathways. These findings identify spliceosome components and RNA processing machinery as crocin-sensitive pathways.