Patel, Aalia2023-11-172023-11-172022https://hdl.handle.net/10539/37030A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Molecular and Cell Biology to the Faculty of Science, University of the Witwatersrand, Johannesburg, 2022Our genomes undergo DNA damage regularly with the most lethal being DNA double-strand breaks (DSBs). Long non-coding RNAs (lncRNAs) have distinct biological functions and play roles in DNA damage response (DDR). We hypothesized that lncRNA expression would be altered in response to NaCl-induced DNA damage and that these effects may be different in cancer and non-cancer cell lines. Exposure of cells to high NaCl concentrations promotes DSBs in DNA regions near the nuclear periphery. These are rapidly repaired after NaCl withdrawal. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays were performed on HEK293 and HT29 cell lines to determine the EC50 for NaCl. For confocal microscopy, cells were treated with NaCl for 6 hrs or 24 hrs followed by 5 min NaCl withdrawal, and Mre11 was detected. Results indicated that meiotic recombination 11 homolog 1 (Mre11) was localized in the cytoplasm in the presence of NaCl and translocated to the nucleus during 5 min NaCl withdrawal in HEK293 cells. Under high NaCl, Mre11 was present in both the nucleus and cytoplasm in HT29 cells. DSB assessment by western blot analysis for gH2AX revealed no significant change in expression in HEK293 and HT29 cells, but slightly lower levels in HT29 suggest DNA repair may occur in the presence of NaCl. PCR and qPCR were performed for lncRNAs p21 associated ncRNA DNA damage activated (PANDA), Antisense non-coding RNA in the INK4 locus (ANRIL), and DNA damage sensitive RNA1 (DDSR1). qPCR data shows that PANDA, ANRIL, and DDSR1 expression is reduced in HEK293 during NaCl treatment, while in HT29s PANDA was upregulated in response to NaCl treatment and NaCl withdrawal, ANRIL was only upregulated after 4 hrs NaCl and in the 6 hr 5 min NaCl withdrawal treatment and DDSR1 expression was significantly higher in the 6 hr treatment only. Whilst hyperosmotic stress was used to induce DNA damage, we have observed that it results in reduced expression of lncRNAs that play roles in DNA repair and cell cycle regulation in a non-cancer cell line, and enhanced expression in a cancer cell line. This suggests that in hypertonic environments the HT29 cells may still be undergoing cell cycle arrest, DNA repair, and proliferation.enGenomesDNA damageCancerDissertation