Temporal transcriptomic profiling identifies core regulators in cytokine-induced gut barrier disruption in differentiated Caco-2 cells

OBJECTIVE: The intestinal epithelial barrier maintains gut homeostasis, and its disruption contributes to inflammatory diseases such as Inflammatory Bowel Diseases (IBD). Although barrier restoration is a promising therapeutic strategy, most current approaches target immune responses rather than epithelial function. Since barrier injury and repair progress through discrete phases, this study aimed to define time-resolved epithelial transcriptional programs and identify candidate regulatory nodes for barrier-focused therapeutic intervention. METHODS: Differentiated Caco-2 epithelial monolayers were used to model cytokine-induced barrier disruption and profiled by time-resolved bulk RNA-seq. Transcriptional dynamics and candidate regulators were identified using pairwise differential expression, likelihood-ratio testing, pathway enrichment, and upstream-regulator inference. Candidate nodes were tested by pharmacologic perturbation, and disease relevance was assessed by qPCR and histology in human IBD colon tissues. RESULTS: Time-resolved profiling revealed dynamic epithelial programs involving the Rho GTPase cycle, sirtuin signaling, non-canonical WNT signaling, and junction/EMT-associated pathways. Upstream-regulator inference highlighted JAK2, TNF, gasdermin D, and β-estradiol, and pharmacologic perturbation of these nodes mitigated cytokine-induced barrier loss. HNF4A emerged as a gut epithelial transcriptional hub regulating targets including SATB2 and SLC26A3. Notably, SLC26A3 was markedly reduced in IBD colon tissues. CONCLUSIONS: This study provides a time-resolved epithelial framework and highlights therapeutic opportunities for barrier-focused interventions.