Falkov, Jemma Lilian2024-10-252024-10-252023-07Falkov, Jemma Lilian. (2023). Expression and Methylation of Peroxidasin in Breast Cancer Cell Lines. [Master's dissertation, University of the Witwatersrand, Johannesburg]. https://hdl.handle.net/10539/41966https://hdl.handle.net/10539/41966A dissertation Submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023.Peroxidasin (PXDN) is a haem-containing extracellular matrix peroxidase protein which forms hypohalous acids in the presence of hydrogen peroxide (H2O2). The predominant role of PXDN is that of a collagen IV crosslinker within the basement membrane. Increased collagen IV deposition has been linked to tissue invasion and metastasis in breast cancer and PXDN has also been shown to assist in the process of epithelial-mesenchymal transition (EMT) in cancer. Various cancer types display dysregulated levels of PXDN expression including breast cancer and this dysregulation has been associated with poor prognosis. This study aimed to investigate whether DNA methylation of the PXDN promoter may be a mechanism through which changes in PXDN expression observed in breast cancer are regulated. Non-invasive MCF-7 and invasive MDA-MB-231 cells were used as models for luminal A and triple negative breast cancer (TNBC) respectively. The HEK-293 cell line was used as a non-cancerous control cell line. DNA methylation levels of the PXDN promoter and PXDN protein expression was investigated in these cell lines through the methods of methylation sensitive PCR (MS PCR) and immunofluorescence microscopy. Relative levels of PXDN expression were determined through immunofluorescence microscopy. Corrected total cell fluorescence (CTCF) analysis of these images revealed the highest PXDN levels to be found within the invasive MDA-MB-231 cell line, which was double that of the MCF-7 cell line. All cell lines were treated with 10 nM β-Oestradiol, which caused an increase in PXDN expression within the MCF-7 and HEK-293 cell lines and a decrease in expression within the MDA-MB-231 cell line to half its untreated value. PXDN was found to be localised in the ECM in all three cell lines. To elucidate the role of DNA methylation, methylation sensitive PCR (MS PCR) was performed on all three cell lines, with four primer pairs spanning a region of 1305 base pairs (bp) within the PXDN promoter. A region of differential methylation was found between the MDA-MB-231 and HEK-293 cell lines between 524 bp and 53 bp upstream of the transcription start site (TSS). This region was unmethylated within the MDA-MB-231 cell line and methylated within the HEK-293 cell line, which correlates with expression differences between these two cell lines and suggests this region could be of regulatory significance. The four primer pairs designed to amplify the PXDN promoter were unable to amplify this region within the MCF-7 cell line. A heterochromatic DNA conformation or a point mutation increasing CpG content creating a thermodynamically ultra-fastened (TUF) region could be the explanation behind this phenomenon, however further research is required to elucidate the mechanism responsible. In conclusion, PXDN shows higher expression in TNBC cells than in luminal A subtype cells. The oestrogen receptor is involved in regulating PXDN expression, however, different mechanisms seem to be at play between the two cell lines. The contribution of CpG methylation to this change in PXDN expression remains unknown, as does the nature of the interaction between the oestrogen receptors and the gene. Further research is required to clarify the mechanisms involved.en©2023 University of the Witwatersrand, Johannesburg. All rights reserved. The copyright in this work vests in the University of the Witwatersrand, Johannesburg. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of University of the Witwatersrand, Johannesburg.DNA methylationTriple negative breast cancerPeroxidasin (PXDN)Hydrogen peroxide (H2O2)Epithelial-mesenchymal transition (EMT)UCTDSDG-3: Good health and well-beingDissertationUniversity of the Witwatersrand, Johannesburg