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

Abstract

Peroxidasin (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.