Browsing by Author "Sebastian, Mistral"

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    Investigating the DNA methylation status of the PXDN and PXDNL promoter regions in OSCC cell lines
    (University of the Witwatersrand, Johannesburg, 2024-06) Sebastian, Mistral; Mavri-Damelin, Demetra
    Background: Oesophageal squamous cell carcinoma (OSCC) is the most prevalent form of oesophageal cancer in South Africa. Aberrant DNA methylation is a well-established epigenetic mechanism involved in various cancers, including OSCC. This study focuses on the DNA methylation status of the peroxidasin (PXDN) and perodixasin like (PXDNL) promoter regions and the expression of PXDN and PXDNL in OSCC cell lines. PXDN consolidates the basement membrane through collagen IV unit oligomerization, influences epithelial-mesenchymal transition and correlates with poor prognosis in various cancers. PXDNL modulates the extracellular matrix (ECM) by antagonising PXDN. Since PXDNL shares domains with PXDN, that allow PXDN to interact with the ECM, it is speculated that PXDNL may possess other ECM modulation roles that require further elucidation. Dysregulated PXDNL expression also correlates with poor cancer prognosis. To date, within the context of South African derived OSCC cell lines, no studies pertaining to the DNA methylation status of the PXDN and PXDNL promoter regions and the expression of PXDN and PXDNL have been carried out. Aim: The aim of this project was to investigate the DNA methylation status of the PXDN and PXDNL promoter regions and observe PXDN and PXDNL expression in the SNO and WHCO5 OSCC cell lines. Methods: PXDN and PXDNL localisation was observed using immunofluorescence microscopy; expression of PXDN and PXDNL was quantified using western blotting and the DNA methylation status of the PXDN and PXDNL promoters was assessed using methylation specific PCR and bisulfite sequencing, respectively. Results: Immunofluorescence microscopy results indicated that both cell lines show varying degrees of PXDN and PXDNL expression. In addition, these results also showed that PXDN and PXDNL localise in the ECM. The western blotting results established that these cell lines express the canonical version of PXDN and possibly a PXDNL isoform (146kDa). Methylation specific PCR has shown that the promoter region of PXDN is differentially methylated across both cell lines. The sequencing results of the bisulfite converted PXDNL promoter region were unsuccessful. Hence, bisulfite sequencing requires further optimisation before the DNA methylation status of the PXDNL promoter region can be determined. Conclusion: This study is the first to show the novel finding that PXDN and PXDNL are expressed in South African derived OSCC cell lines. Within the context of OSCC, further investigation is warranted in order to elucidate the underlying mechanisms that these proteins play a role in. In addition, further study may determine whether a correlation exists between PXDN and PXDNL promoter methylation, protein expression as well as prognosis and whether these aspects should serve as novel markers for diagnosis and therapy. This may subsequently lead to increased OSCC patient survival rates by contributing to early diagnosis of OSCC and efficacious targeted therapeutic intervention.