Abstract 1936 Glucocorticoid Receptor Mediated DNA Repair Mechanisms in Human Corneal Epithelial Cells

The cornea is a transparent, avascular tissue located on the anterior portion of the eye. Its primary function is to serve as a protective barrier to the eye from structural damage, infection, and environmental factors such as UV rays. Out of the three classifications of UV rays, the earth's atmosphere absorbs the most harmful, UVC, allowing UVB and UVA rays to pass through and induce DNA breaks and mutations to our barrier tissues: skin and eyes. While both UVB and UVA cause damage, UVB is more severe and is linked to causing DNA mutations leading to cancer in the skin. In the eye, UVB is prevented from reaching the retina but, consequently, causes severe damage to the cornea, which aids in the protection of the rest of the eye by absorbing most of the UVB. DNA damage in the cornea occurs through multiple cellular pathways, but the glucocorticoid receptor (GR) DNA repair mechanisms are less known. GR is a transcriptional factor and nuclear receptor capable of regulating the transcription of genes involved in numerous body functions such as cognition, blood pressure, and inflammatory response. GR-mediated DNA repair is shown in other organs, but less is understood about its role in the cornea. Our objective is to investigate how GR is involved in DNA repair using UVB-treated corneal cells by employing gene expression assays and slot blot technique. Human Corneal Epithelial Cells (HCET) were transfected through a four-day process with either NTC siRNA (non-target control) or GR siRNA to knock down GR expression. Cells were then treated with 50J/m2 UVB and incubated for their respective time points of 0 hr, 1 hr, and 6 hr and untreated (UT) before being collected for RT-qPCR analysis of DNA repair genes. When choosing which DNA repair genes to investigate, PARP1, HMOX1, and SOD1 were selected due to their prevalence in the DNA repair process as stated by prior literature. After analysis of the selected genes, we demonstrated that the loss of GR in human corneal epithelial cells (GRKD) led to the upregulation of all of our DNA repair genes. The upregulation of these repair genes in GRKD samples implies that GR inhibits the expression of the chosen genes. To further investigate GR mediation of DNA repair, a slot blot assay will be performed to analyze the clearance of DNA mutations, specifically cyclobutane pyrimidine dimers (CPD), relative to time point 0 hr in HCET cells. Analysis of CPD clearance over UT, 0 hr, 1 hr, and 6 hr, will provide more detailed information regarding DNA mutations, leading to a better understanding of the mechanism of GR in the DNA repair process in the cornea.