Glucocorticoid Receptor Mediated DNA Repair Mechanisms in Human Corneal Epithelial Cells
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Issue Date
2024-04-29
Authors
O'Neill, Erin
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First Reader
Kadmiel, Mahita
Additional Readers
Nelson, Margaret K.
Keywords
Glucocorticoid Receptor , DNA repair , Cornea
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Abstract
Glucocorticoids are an anti-inflammatory treatment used for a plethora of diseases but can cause negative side effects. More research is needed to understand the mechanism through which glucocorticoids work to allow for healthy use, specifically within the eye. 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. UVB rays induce DNA mutations leading to cancer in the skin. In the eye, UVB causes sun damage to the cornea, which 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. 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 UT (untreated), 0 hour, 1 hour, and 6 hours before being collected for qRT-PCR 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 and mediation by GR as stated by prior literature. After analysis of the selected genes, we demonstrated that the loss of GR in HCET cells (GRKD) led to the upregulation of 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 was run to analyze the clearance of cyclobutane pyrimidine dimer mutations relative to time point 0 hour in HCET cells. Analysis of CPD clearance over UT, 0 hour, 1 hour, and 6 hours, demonstrated inconclusive data due to presence of CPD mutations in UT samples. Further research is needed to determine the effects of GRKD on DNA mutation clearance ability within HCET cells. Analysis of DNA repair genes in HCET cells provides a better understanding of the mechanism of GR in the DNA repair process in the cornea where GR inhibits the expression of DNA repair genes PARP1, HMOX1, and SOD1, therefore furthering research on healthy glucocorticoid use.
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Biology
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Biology
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Biology, 2024