Impact of pEGFP mediated ING4 gene on growth of glioma U251 cells and its potential molecular mechanism

Abstract

To investigate the impact of the inhibitor of growth family 4 (ING4) on growth of glioma cells (U251 cells) and its potential molecular mechanism, total RNA was extracted from the embryonic tissues and ING4 was amplified by RT-PCR and cloned into pEGFP-C2 vector. U251 cells were transfected with eukaryotic expression vector pEGFP-ING4 mediated by cationic polymers polyethylenimine (PEI). Flow cytometry and G418 were used to screen the cells successfully transfected with pEGFP-ING4. PEGFP-ING4 group, pEGFP group and blank control group (without transfection) were included in this study. Morphological examination, MTT assay and Hochest staining were employed to detect the proliferation and apoptosis of U251 cells. RT-PCR, immunohistochemitry and western blot were recruited to determine the mRNA and protein expression of ING4, respectively. ELISA was performed to measure the VEGF level in the supernatant of U251 cells. Sequencing of pEGFP-ING4 showed sequence correctness and the tranfection efficiency was 84% for pEGFP-ING4 and 82% for pEGFP. Results from RT-PCR, immunohistochemistry and western blot revealed significantly increased ING4 expression. When compared with the pEGFP group and blank group, the growth inhibition rate and apoptotic rate 72 h after transfection in the pEGFPING4 group were significantly higher (P 0.05). ING4 can be highly expressed in the pEGFP-ING4 group and remarkably suppress the growth of U251 cells through inducing apoptosis but not suppressing VEGF expression. Key words: Inhibitor of growth family member 4, glioma, angiogenesis, vascular endothelial growth factor.