Investigating The Oncogenic Function of Brain Specific CYP4X1 Gene in Glioblastoma Cancer Cells Using RNA-i Mediated Gene Repression Strategy

Date

2023-6-21

Author

DURUKAN, Özlem

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Brain tumor cells show an aberrant blood vessel formation which makes angiogenesis a potential target for brain tumor treatment. Arachidonic acid and its endogenous amide, arachidonoyl ethanol amide (anandamide) are known to have a regulatory impact on cancer cell angiogenesis. Therefore, enzymes which are responsible for the metabolism of these endogenous compounds, have been frequently investigated as possible angiogenic targets. CYP4X1 is a newly discovered member of Cytochrome P450 enzyme family with unknown specific function. Since, it is specifically expressed in brain and responsible for the metabolism of anandamide, makes CYP4X1 as a new plausible target for regulation of brain tumor angiogenesis. This study investigates the potential relevance of the CYP4X1 gene in brain tumor angiogenesis, using T98-G human glioblastoma multiforme cell line. CYP4X1 expression was knocked down via RNA interference (RNAi) system using Silencer Select pre-designed three independent siRNA mix, targeting CYP4X1 mRNA and scramble (negative) control siRNA. In addition, to understand the possible angiogenic effect of the CYP4X1 gene VEGF-A gene and protein expressions in T98-G cells were investigated by using q-RT PCR and Western blot techniques, respectively. Our results indicate that although VEGF-A gene expressions were significantly downregulated, protein expressions didn’t change after CYP4X1 gene repression in T98-G cells. Accordingly, VEGF-A secretion which was measured with sandwich ELISA method from cell free supernatants, was significantly reduced in siRNA transfected group compared to the control group. Human umbilical endothelial vein (HUVEC) cell line was used to further investigate the effect of CYPX1 in vascular endothelial cells. No statistically significant difference was observed in HUVEC cell proliferations among the groups. In addition, in vitro angiogenesis assay was constructed by using HUVEC cells on Matrigel, and the tube length, loop formation and branching points system were evaluated by using inverted light microscope. All parameters were seemed to be reduced under CYP4X1 repression. Finally, alterations in peroxisome proliferator-activated receptor (PPARα) protein expressions were determined by using western blot technique as a possible mechanism behind CYP4X1 mediated angiogenesis. No statistically significant difference in PPARα protein expression among control and CYP4X1 siRNA treated groups was observed. Overall, the results obtained in here indicate that CYP4X1 gene is a promising alternative target for brain tumor angiogenesis. To the best of our knowledge, this thesis work is the first that investigates the possible role of CYP4X1 gene in brain tumor angiogenesis in human cell lines with siRNA mediated gene repression strategy.

Subject Keywords

CYP4X1, RNAi mediated gene silencing, siRNA, Brain tumor, Angiogenesis

URI

https://hdl.handle.net/11511/104491

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Graduate School of Natural and Applied Sciences, Thesis