Differential gene expression and gene-set enrichment analysis in Caco-2 monolayers during a 30-day timeline with Dexamethasone exposure A data modeling approach to understanding culture age as co-variate for differential expression in a non-renewing epithelial monolayer using a gene ontology-defined 250-plex Nanostring probe panel

Glucocorticoid hormones affect gene expression via activation of glucocorticoid receptor NR3C1, causing modulation of inflammation and autoimmune activation. The glucocorticoid Dexamethasone is an important pharmaceutical for the treatment of colitis and other inflammatory bowel diseases. While suppressive effects of glucocorticoids on activated immune cells is significant, their effects upon epithelial cells are less well studied. Previous research shows that the effects of Dexamethasone treatment on polarized Caco-2 cell layer permeability is delayed for >10 treatment days (as measured by transepithelial electrical resistance). In vivo intestinal epithelial cells turn over every 3-5 days; we therefore hypothesized that culture age may produce marked effects on gene expression, potentially acting as a confounding variable. To investigate this issue, we cultured polarized Caco-2 monolayers during a 30-day timecourse with similar to 15 days of continuous Dexamethasone exposure. We collected samples during the timecourse and tested differential expression using a 250-plex gene expression panel and Nanostring nCounter (R) system. Our custom panel was selectively enriched for KEGG annotations for tight-junction, actin cytoskeleton regulation, and colorectal cancer-associated genes, allowing for focused gene ontology-based pathway enrichment analyses. To test for confounding effects of time and Dexamethasone variables, we used the Nanostring nSolver differential expression data model which includes a mixturenegative binomial modelwith optimization. We identified a time-associated EMT-like signature with differential expression seen in important actomyosin cytoskeleton, tight junction, integrin, and cell cycle pathway genes. Dexamethasone treatment resulted in a subtle yet significant counter-signal showing suppression of actomyosin genes and differential expression of various growth factor receptors.