Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes

Abstract

Epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF), and their receptors, epithelial growth factor receptor (EGFR) and bFGF receptor (bFGFR), are frequently overexpressed in high-grade gliomas. In the present study, the EGF and bFGF levels in U251 glioblastoma cell culture supernatants were determined by ELISA, and enhanced green fluorescent protein (EGFP)-labeled recombinant lentiviral expression vectors with small interfering RNA targeting the EGFR and bFGFR genes were constructed. The mRNA expression levels of EGFR, bFGFR, cluster of differentiation (CD)133, glial fibrillary acidic protein (GFAP), tubulin-β3 (TUBB3) and myelin basic protein (MBP) were determined using quantitative polymerase chain reactions in U251 cells prior to and following silencing of the EGFR and/or bFGFR genes. Prior to silencing, the U251 cells secreted EGF and bFGF, and expressed EGFR, bFGFR, CD133, GFAP, TUBB3 and MBP mRNA. Subsequent to silencing the EGFR and/or bFGFR gene, CD133 mRNA expression decreased and GFAP and TUBB3 mRNA expression increased. Silencing the EGFR and FGFR genes acted synergistically to downregulate CD133 expression. The downregulation of CD133 mRNA expression and the upregulation of GFAP and TUBB3 mRNA expression were not significantly different when blocking the EGFR and FGFR pathways. These results indicate that autocrine or paracrine EGF and/or FGF mechanisms exist in U251 cells. Knocking down the EGFR and/or FGFR genes downregulates CD133 mRNA expression and facilitates glial and neuronal differentiation in U251 cells.