Investigating the role of the FOXL2 C134W mutation on the pathogenesis of granulosa cell tumours

Regora, Samantha 2019, Investigating the role of the FOXL2 C134W mutation on the pathogenesis of granulosa cell tumours, B.Science (Hons) thesis, School of Life and Environmental Sciences, Deakin University.

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Title Investigating the role of the FOXL2 C134W mutation on the pathogenesis of granulosa cell tumours
Author Regora, Samantha
Institution Deakin University
School School of Life and Environmental Sciences
Faculty Faculty of Science, Engineering and Built Environment
Degree type Honours
Degree name B.Science (Hons)
Thesis advisor Chu, Simon
La Fontaine, SharonORCID iD for La Fontaine, Sharon orcid.org/0000-0002-9948-074X
Date submitted 2019-12-09
Keyword(s) ovarian cancer
genetics
mutation
FOXL2
Summary Granulosa cell tumours (GCT) arise from the ovarian sex cord-stroma and comprise a unique subset of malignant ovarian tumours. GCT are an indolent tumour with an unusual propensity for late recurrence. Whilst initial prognosis is favourable, one third of women will experience an aggressive or relapsed tumour, with a mortality rate of ~80%. GCT main treatment is surgery, adjuvant therapies such as hormonal therapy and chemotherapy are met with limited efficacy. A pathognomonic mutation in the FOXL2 gene of GCT is well described and may be a potential target for novel therapeutics, however the pathogenesis of this C134W mutation is not yet fully understood. Thus, the present study aimed to investigate the role of C134W on the pathogenesis of GCT for future therapeutic potential. CRISPR was used to introduce or revert the C134W mutation in an immortalised granulosa cell line and a GCT derived cell line to allow investigation of the addition or removal of the pathognomonic mutation on cellular functions. The guide RNA target for the FOXL2 gene was designed immediately upstream of the 5’ untranslated region of the gene and successfully ligated with a Cas9 plasmid. FOXL2 homology arms were successfully cloned into a recombinant vector HR110PA-1 to act as the donor fragment for CRISPR Cas9/gRNA co-transfection using homology directed repair pathway to be activated. Site directed mutagenesis of the wild-type sequence was required to convert the sequence to C143W into the FOXL2 gene of the vector donor fragment. Whilst unsuccessful in the HR110PA-1 vector, C134W was successfully introduced into a FOXL2-GFP plasmid which will be utilised in future overexpression studies. Further work is required to develop the CRISPR system for insertion or removal of C134W into various cell lines. Once these new cell lines are designed, functional assays will be utilised as part of a 6-way comparison study model centred around proliferation, apoptosis and steroidogenesis, which are potentially impacted by the FOXL2 mutation. This research project has provided a strong foundation for future functional studies with the translational potential for improving GCT treatments and patient outcomes.
Language eng
Indigenous content off
Field of Research 1112 Oncology and Carcinogenesis
Description of original 83 p.
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Persistent URL http://hdl.handle.net/10536/DRO/DU:30133340

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