Georgiadi, Eleni (2022) Periostin splice variants in pancreatic cancer. (PhD thesis), Kingston University, .
Abstract
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid tumours, with 5-year survival of less than 5%. The aggressive nature of the tumour, the tumour-supportive environment (stroma), delayed diagnosis and poor chemotherapy response lead to poor patient prognosis. The tumour-stroma is a dynamic network of immune, mesenchymal, and endothelial stromal cells surrounded by an extracellular matrix rich in matricellular and architectural proteins. Periostin (POSTN), a matricellular protein primarily secreted by stromal cells, is often associated with cell proliferation and migration. POSTN overexpression has been observed in many tumour types, however downregulation in bladder carcinoma rises some questions. POSTN is an alternatively sliced gene, that encodes multiple different isoforms, whose function in PDAC is still unknown. The aim of this study is to isolate and clone these isoforms in order to produce recombinant proteins for further investigation. Methods: The transcript variants were extracted from PS1 cells (a pancreatic stromal cell line), converted to cDNA and amplified by RT-PCR. The synthesised DNA was ligated with plasmid vectors and cloned by traditional cloning into E.coli cells. Single colonies were sequenced and analyses, determining the transcript variant being cloned. The plasmids ligated with the variants of interest were isolated and transfected into PS1 cells. The recombinant proteins synthesised by the PS1 cells were then purified. Results: Multiple isoforms of POSTN were cloned in E.coli. Sequencing analysis revealed the exons present in each sample of the cloned colonies and the isoforms were identified. The plasmids of interest were successfully extracted from the colonies by using purification technique appropriate for subsequent mammalian cell transfection. Substantial progress was made in producing recombinant POSTN proteins. Conclusion: In this study the isolation of three cloned POSTN transcript variants were achieved. These variants are POSTN-202, 209 and 201-b.
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