Résumé
Background − Glioblastoma (GBM) is a highly aggressive glioma with an average survival of 15 months. The current standard of care is a multimodal approach based on surgery, concomitant chemotherapy and radiotherapy as well as adjuvant chemotherapy. The intratumour heterogeneity poses as the greatest challenge in developing a better treatment as it makes this cancer highly resistant to treatment. Next-generation sequencing (NGS) technologies have improved the understanding of complex diseases such as cancer. Furthermore, the discovery of genome-editing techniques such as CRISPR-Cas endonucleases have provided scientists with the right tools to target specific genes within a given cell genome and manipulate their expression. Previously to the start of this project, a genome-wide CRISPR-Cas9 knockout (GeCKO) screening was performed on a GBM cell line upon a combined exposure to X-ray irradiation and temozolomide (TMZ) to determine which genes are implicated in the resistance to GBM treatment. The results suggested that TRAPPC genes, which participate in intracellular trafficking, may have a role in the sensitivity to GBM treatment.Aim – In this study, the aim was to validate whether some of the TRAPPC genes play a role in the GBM sensitivity to treatment.
Methodology –The project was divided into two strategies: the knockout and the knockdown of TRAPPC1 gene. For the knockout of TRAPPC1 gene, three TRAPPC1-targeting-single-guide RNAs (sgRNA) were designed and cloned into a plasmid containing the CRISPR-cas9 endonuclease. For the knockdown of TRAPPC1 gene, three targeting TRAPPC1-shRNAs plasmids were designed. Using lentivirus as the gene-therapy vector system, the plasmids were delivered into the U87 cells. The validation of the knockout/knockdown was done at mRNA and protein levels. Additionally, a proliferation assay was performed. Then, TRAPPC1-knockdown GBM cells were treated with a combination of radiotherapy and TMZ and a clonogenic assay was performed to study the role of TRAPPC1 gene in the sensitivity to treatment.
Results – The knockout of TRAPPC1 was validated at mRNA and protein levels for one of the TRAPPC1-targeting sgRNA. The knockdown of TRAPPC1 was validated for one of the TRAPPC1-targeting shRNA at mRNA level. The validation at protein level was not successful. A decrease in cell proliferation was observed with the knockout and knockdown of TRAPPC1. After TMZ and X-ray treatment, TRAPPC1-knockdown cells seem to have a slight higher clonogenicity than controls.
Conclusions – TRAPPC1 appears to be involved in cell proliferation and thus, behaves like an essential gene. The implication of TRAPP genes in the sensitivity of GBM to treatment was not demonstrated and thus, further research needs to be done.
| la date de réponse | 26 août 2022 |
|---|---|
| langue originale | Anglais |
| L'institution diplômante |
|
| Superviseur | Carine Michiels (Promoteur) & Jia-Wei Chen (Copromoteur) |