Glioblastoma is a malignant form of brain cancer. This type of tumour is resistant to medical treatment and often yields a poor prognosis. It is important to understand the molecular basis of glioblastoma for developing new therapies. This study used U87 human glioblastoma cells for the investigation. The glioma cell line displayed the hallmarks of a tumour cell and corresponded on various cellular functions that are regulated by Rho family GTPases. This study intended to analyse the role of one member from this family - RhoE in U87 cells. RNA interference (RNAi) method was applied to knock down RhoE. The result showed that the loss of RhoE expression is associated with the decrease in cell cycle progression and increase apoptosis in U87 cells. Endogenous RhoE was found localised to the nucleus in U87 cells. RhoE is known can interact with serine/threonine kinases called ROCK1 and PKC. The application of RNAi that knocks down ROCK1 has led to the stabilisation of RhoE expression in U87 cells. Y27632 was used to inhibit ROCK1 whereas Gӧ6976 blocked the function of PKC. Both pharmacological inhibitors prevent individual interaction with RhoE. The activation of novel PKC by PMA exhibit an increased the upper band of RhoE (29kD) and it remains more in the nucleus consistent with the active conventional PKC during ROCK1 inhibition. A co-immunoprecipitation (Co-IP) technique was used to pull down FLAG-RhoE from the transfected U87 cells with wild-type construct. As a result, the translocation of RhoE to the nucleus was facilitated by the interaction with importin (Imp). A bioinformatics study on protein-protein interactions using DAVID on mass spectrometry data elucidated that RhoE could be indirectly bound to nuclear proteins. In conclusion, RhoE plays a pivotal role in regulating glioblastoma at the post-translational level.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:768309 |
| Date | January 2018 |
| Creators | Mohd Zahari, Maihafizah Binti |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/8809/ |
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