Differential gene expression and gene-set enrichment analysis in Caco-2 monolayers during a 30-day timeline with Dexamethasone exposure

ABSTRACT

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 ~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® 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.

KEYWORDS:

• Epithelial homeostasis
• epithelial barrier function
• epithelial permeability
• Caco-2
• Dexamethasone
• glucocorticoid
• NanoString
• TEER
• RhoGTPase
• tight junction

Abbreviations

Table

Acknowledgments

Robinson, Turkington, Kenea were funded through NIH IRTA fellowships. The research was funded to Wendy A. Henderson through 1Z1ANR000018-01-8. Authors acknowledge Ethan Tyler of the NIH Medical Arts Division for his graphic design work on Figure 1, and Dr Greg Gonye for Nanostring nCounter training.

Author Contributions

Robinson designed the experiments, conceived and designed the custom Nanostring codeset, performed cell cultures, collected TEER data, performed RNA extractions, performed Nanostring protocols and operated the nCounter instrument for each replicate, on-site at the Henderson Lab in the NIH Clinical Center. Robinson performed statistical and bioinformatic analyses using the R-based nSolver™ 4.0 and Advanced Analysis 2.0 software packages, with manual checking of results, and TEER data using R/R-Studio. Robinson wrote the text, provided revisions, and produced figures (see acknowledgments for additional Figures 1 and 2 contributions).

Turkington performed cell culture tasks, RNA extraction, and operation of Nanostring nCounter®.

Abey contributed to development of cell culture workflow and methods, contributed to design of experiments, contributed to Nanostring codeset design and content, and provided comments for the text.

Kenea performed additional cell culture tasks.

Henderson contributed to design of experiments, contributed to the Nanostring codeset design and content, supervised and administered the research, edited and provided revisions to the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplementary data for this article can be accessed here

Additional information

Funding

This work was supported by the National Institutes of Health [1Z1ANR000018-01-8].