Publication: The effects of EPHA2 inhibition on VEGF, VEGFR-1 and VEGFR-2 in human malignant glioma cells
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Date
2018-04
Authors
Baharuddin, Wan Noor Ainun
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Abstract
Brain tumor “glioblastoma multiforme (GBM)” is assigned under fourth
pathologic grades (grade IV) of astrocytic tumor. Despite various advances in the
treatment modalities, median survival rate of GBM patients remains low between 12
to 14 months with a poor clinical prognosis. Human EphA2 gene plays an important
role in regulating angiogenesis and known to be over expressed in GBM tumors. The
elevated trend of EphA2 was associated to the poor prognosis of GBM patients, shorter
survival rate and recurrence of the disease. This study aims to investigate the roles of
EphA2 in angiogenesis signaling pathway governed by VEGF, VEGFR-1 and
VEGFR-2. In this study, two types of cell lines derived from different origins were
utilized. U-87MG cells were isolated from a patient with GBM type 4 diagnosis,
whereas DBTRG-05MG was isolated from a GBM type 4 patient who underwent
chemotherapy and radiotherapy. The effects of EphA2 gene silencing following
transfection of small interfering RNA (siRNA) on the gene and protein expression of
EphA2 and tumor angiogenesis related markers; VEGF, VEGFR-1 and VEGFR-2
were evaluated by using quantitative Real Time-PCR and Western blot analysis. This
study showed downregulation of EphA2 mRNA (p=0.0061) had significantly
downregulated VEGF (p=0.0123) and VEGFR-2 (p=0.0299) mRNA expression in U-
87MG cells. Consequently, at protein level there was no difference in VEGF
expression (0.98-fold relative to 1-fold of non-transfected group), but decrement of
VEGFR-2 expression by 0.51-fold was observed. As for DBTRG-05MG cells,
downregulation of EphA2 mRNA (p=0.0137) had led to an increasing trend of VEGF
mRNA expression (p=0.2131), but decreasing trend of VEGFR-2 (p=0.1453). Both
differences were not statistically significant. At protein translational level, there was
no difference of VEGF protein expression (0.92-fold relative to 1-fold of nontransfected
group), but reducing VEGFR-2 protein by 0.61-fold decrease. This study
also had shown that there was no VEGFR-1 gene and protein expression in both U-
87MG and DBTRG-05MG malignant glioma cells. Therefore, it was discovered that
EphA2 plays an important role in VEGF/VEGFR-2 signaling in GBM cells. In
conclusion, this study demonstrated that silencing EphA2 mRNA expression by
siRNA led to the downregulation of gene and protein expression of VEGFR-2 without
affecting VEGF secretion in malignant glioma cells. Interestingly, this study has
successfully elucidated that EphA2 plays important roles in mediating angiogenesis
through VEGFR-2 signaling.
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Keywords
Glioma