3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Peroxidasin is a novel target of NRF2 and influences epithelial-to-mesenchymal transition during oxidative stress(2018) Hanmer, Kerry LeighPeroxidasin (PXDN) is a member of the haem-peroxidase protein family that contains a conserved peroxidase domain and additional extracellular matrix (ECM) protein binding domains. The functions of PXDN are still being identified although it has been shown to i) oxidise molecules by utilising hydrogen peroxide (H2O2), ii) facilitate formation of dityrosine cross-links and iii) catalyse sulfilimine bond formation within the basement membrane. PXDN expression is altered in various diseases such as cancer and therefore it is important to determine the regulators and functions of PXDN so as to identify new therapeutic targets. Since PXDN utilises H2O2, and previous studies have shown PXDN expression changes in response to H2O2, we hypothesised that the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor is involved in PXDN regulation. Western blot analyses and immunofluorescence microscopy confirmed an increase in Nrf2 and PXDN expression during oxidative stress and upon treatment with Nrf2-specific inducers. Chromatin immunoprecipitation confirmed binding of Nrf2 to the PXDN promoter and luciferase assays indicated that Nrf2 binding to the PXDN promoter increases luciferase reporter gene expression. Mutagenesis of the potential Nrf2 binding site within the PXDN promoter significantly decreased gene expression thus confirming that Nrf2 regulates PXDN. A role for PXDN in cancer was then investigated and it was hypothesised that PXDN is potentially involved in epithelial-to-mesenchymal transition (EMT) during oxidative stress. Knockdown of PXDN expression (siRNA-mediated) was quantified by ELISA and preliminary assays were performed on wild type HeLa and SiHa cervical carcinoma cells in comparison to knockdown cells. PXDN knockdown in both cell lines resulted in decreased cell attachment. In HeLa cells, oxidative stress resulted in increased migration but PXDN knockdown abolished this effect. Oxidative stress in HeLa cells decreased invasion with PXDN knockdown resulting in a further decrease. PXDN knockdown in SiHa cells decreased cell migration and SiHa cells under oxidative stress exhibited increased invasion, which was reversed by PXDN knockdown. Reduced PXDN expression decreased the size and formation of 3D spheroids in both cell lines. In summary, Nrf2 binds the sequence, TGAATCTGGC, within the PXDN promoter and increases PXDN expression. Our preliminary findings also implicate PXDN in redox related EMT since PXDN knockdown caused significant alterations to key EMT features.Item An investigation into the regulation of peroxidasin gene expression by the SNAI1 transcription factor and its involvement in epithelial-to-mesenchymal transition in human cervical carcinoma cell lines(2018) Moleya, Boitumelo NonhlanhlaPeroxidasin (PXDN) is a unique member of the peroxidase - cyclooxygenase superfamily and differs from its counterparts in that in addition to an enzymatic peroxidase domain, PXDN has protein-binding domains characteristic of proteins found in the extracellular matrix. PXDN is integral to basement membrane consolidation and catalyses sulfilimine bonds in collagen IV and covalent cross-linking of dityrosine. PXDN also has microbicidal activity in plasma through the generation of hypochlorous acid and may also catalyse peroxidative reactions intracellularly. Additionally, PXDN is involved in processes where epithelial-to-mesenchymal transition (EMT) takes place, namely fibrosis, development and cancer. In cancer, PXDN has been found to be aberrantly expressed in a colorectal cancer cell line undergoing p53-dependent apoptosis, in malignant primary glial and metastatic brain tumours; and was identified as a candidate tumour suppressor gene silenced by DNA methylation in patients diagnosed with acute myeloid leukemias. PXDN has also been implicated in tumour invasion of choriocarcinoma and melanoma cells. Considering PXDN expression can be modified by transforming growth factor β1 (TGF-β1), a major signaling molecule involved in the initiation of EMT, in this study we aimed to investigate the role of PXDN in TGF-β1-induced EMT in human cervical cancer cells. This included i) identifying whether Snai1, the main transcription factor involved in EMT, regulates the expression of PXDN; and ii) whether PXDN contributes to the various aspects of EMT in cervical carcinoma cell lines. During TGF-β1-induced EMT, PXDN expression decreased in HeLa and SiHa cells, with concomitant increases in Snai1 and vimentin, and decrease in E-cadherin, as quantified by western blotting and visualised by immunofluorescence microscopy. We showed that TGF-β1 induced Snai1 binding to the PXDN promoter, as assessed by chromatin immunoprecipitation-PCR, and significantly repressed luciferase reporter gene expression, as did Snai1 over- expression. We also found that knocking down PXDN expression decreases proliferation, attachment and invasion of HeLa and SiHa cells and increases the migration rate of HeLa cells but decreases that of SiHa cells. In summary, our findings show that Snai1 mediates repression of PXDN and consolidates a role for this ECM-modifier during EMT, in particular during proliferation, attachment and migration of the cervical carcinoma cells, HeLa and SiHa.