TRANSCRIPTOMIC ANALYSES REVEAL DISTINCT RESPONSES OF DIFFERENTIATED AND POORLY-DIFFERENTIATED INTESTINAL EPITHELIAL CELLS TO LIPID PEROXIDES

Abstract

Lipid peroxides (LOOHs) abound in processed food and have been implicated in the pathology of diverse diseases including gut, cardiovascular, and cancer diseases. However, the molecular mechanisms by which LOOHs contribute to disease have not been fully characterized. Caco-2 cells have been widely used to model human intestinal epithelium in metabolic studies. As differentiated (Diff) and poorly-differentiated (PDiff) Caco-2 cells represent good models of human enterocytes and intestinal tumor cells, respectively, we investigated the cellular response of Diff and PDiff Caco-2 cells to the most common dietary LOOH, 13- hydroperoxyoctadecadienoic acid (13-HPODE), in terms of differential gene expression, gene ontology and pathway analysis using transcriptomic profiling. This dissertation demonstrates the work conducted on Diff and PDiff Caco-2 cells in order to understand how LOOHs might contribute to disease in the intestinal epithelium. We also compare between the responses of Diff and PDiff cells to LOOHs as Diff cells resemble mature-like enterocytes (have brush borders) and PDiff cells model intestinal cancer cells or crypt cells (lack brush borders). To characterize gene expression and pathway dysregulation upon exposure to peroxidized linoleic acid, we incubated Diff and PDiff intestinal epithelial cells (Caco-2) with 100μM of 13- hydroperoxyoctadecadienoic acid (13-HPODE), linoleic acid (LA) or hydrogen peroxide (H2O2) for 24h. Total RNA was extracted for library preparation and Illumina HiSeq sequencing. This dissertation demonstrates that the Diff and PDiff Caco-2 cells, which differ in their phenotype, behavior and gene expression, show significant differences in their response to the most common dietary lipid peroxide, 13-HPODE, although some similarities in the enriched processes of both cell types were observed. Both cell types showed enrichment of PPAR iv signaling, cytochrome P450, oxidative phosphorylation and membrane transporters which support previous studies reported the effect of 13-HPODE on these processes. In addition, 13- HPODE treatment had a significant effect on steroid hormone biosynthesis, RNA processing and ribosome biogenesis in both types of cells. On the other hand, Diff cells showed enrichment of bile conjugation upon 13-HPODE treatment which could provide a link between 13-HPODE’s detergent activity and cell phenotype. The significant impact of 13-HPODE on cell cycle and DNA replication/repair in Diff cells might indicate an effect on cellular differentiation and apoptosis. In PDiff cells, more defense mechanisms were triggered by 13-HPODE than in Diff cells. In addition to cytochrome P450, retinol metabolism and peroxisomal pathway were enriched in PDiff cells indicating a stronger defense mechanism was triggered in PDiff than Diff cells. Moreover, phospholipid biosynthesis, amino acid and glycogen metabolism enrichment in PDiff cells, but not in Diff cells, upon 13-HPODE treatment was an indication of tumorigenic environment and malignant transformation. This research using Caco-2 cells provides insights into the physiological changes that might occur in the intestinal epithelial cells upon exposure to 13-HPODE and the possible mechanisms by which it contributes to disease development or progression in intestinal epithelium. Our results also support that Diff and PDiff Caco-2 cells differ in their response to 13-HPODE.