Investigation into the role of the epigenetic target CBX2 in Glioblastoma

Beattie, Hannah

Abstract

Glioblastoma (GBM) has been identified as an extremely difficult tumour to treat owing to issues of heterogeneity, as well as the aggressive nature of the tumours’ growth. Despite current treatments using a combination of methods, treatment resistance contributes to their eventual failing. Therefore, alternative treatment methods are being explored. One such approach includes exploiting epigenetic targeting of key histone modifications. This investigation focusses on the Chromobox domain 2 (CBX2) protein, which has been shown to be overexpressed in several tumour types, including GBM. CBX2 is involved in histone modification, with it being hypothesised that the downstream effects of CBX2’s involvement include silencing of tumour suppressor genes.
This study used both 2D and 3D models to evaluate the role of CBX2 within two GBM cell lines, U-87 MG and SNB-19. RNA interference, through siRNA knockdown, was used to silence CBX2 gene expression in a transient manner. Transfection of plasmids containing full length and chromodomain-depleted CBX2 were also used to cause overexpression of the molecule. The effects of changes to the CBX2 messenger RNA (mRNA) were evaluated using western blotting and RT-qPCR, so as to analyse the change in CBX2 expression at the protein and mRNA level, respectively. An additional first look at the differences of CBX2 expression within a static vs. continuous perfusion microfluidic device also took place, providing a starting point for future investigation of GBM and CBX2, and potentially any other gene of interest, within a more physiologically relevant model.
Key observations included a reduction in cell number within the U-87 MG cell line following the transfection of siCBX2. At the mRNA level, CBX2 expression within SNB-19 was also seen to be significantly reduced, compared to the siScr control, following transfection of siCBX2. CBX2 overexpression altered the expression of CBX2
detected at the protein level, with western blots showing a molecule at 72kDa for the full length CBX2 plasmid and approximately 66kDa for the chromodomain-depleted CBX2 transcript, compared with the 52kDa molecule that was consistently detected in both GBM and breast cancer cell lines. Microfluidic experiments proved successful in
maintaining spheroids on-chip for at least 96 hours, with no significant change in CBX2 expression observed between static and dynamic models.
In the future, it is proposed that the microfluidic devices can be used with patient-biopsies so that a personalised response to therapy, standard or alternative, can be assessed in a way that helps plan treatment. Furthermore, the 3D models could be developed to incorporate more components and be used to better understand GBM biology and develop additional treatment strategies.

Citation

Beattie, H. Investigation into the role of the epigenetic target CBX2 in Glioblastoma. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4866108

• Thesis Type: Thesis
• Deposit Date: Oct 15, 2024
• Publicly Available Date: Oct 25, 2024
• Keywords: Biomedical sciences
• Public URL: https://hull-repository.worktribe.com/output/4866108
• Additional Information: Centre for Biomedicine; University of Hull
• Award Date: Apr 17, 2024