Inhibitory effect of antisense EGFR RNA on the growth of U251 glioma cells

Objective To investigate the inhibitory effect of antisense targeting EGFR on the growth of U251 glioma cells in vivo and in vitro.Methods Antisense EGFR construct p-anti-hEGFR in pcDNA3 was transfected into human malignant glioma U251 cells as mediated by Lipofectamine.Reverse transcript polymerase chain reaction (RT-PCR) and Western blot were rendered to detect the EGFR expression.The proliferative activities of the tumor cells were measured by flow cytometry and in vitro growth on matrigel matrix.Transwell-Matrigel method was used to evaluate the invasive activity in vitro.P-anti-hEGFR and pcDNA3 were injected intratumorally into established subcultaneous U251 glioma in nude mice mediated by lipofectamine,respectively.During observation period of 30 days,tumor volume was measured every 3 days and the tumor mass was taken out at the 30th day.The EGFR expression as well as GFAP was examined by immunohistochemical staining.Results Lipofectamin- mediated antisense EGFR RNA dramatically down-regulated its expression to 85% in U251 glioma cells.Decreased EGFR expression in antisense EGFR RNA transfected cells was accompanied by 12.5% to 13.2% decrease in U251 cells in S phase and 11.9% to 13.4% increase in G_2/M phase.In cell growth matrigel matrix showed normal cell appearence in parental cells and in pcDNA3 transfected cells.While the cells transfected with EGFR antisense RNA were detached from the matrix or grew in a scattered clustering patterns,implicating poor cell growth activities.The invasive activity was significantly dropped down 37.62% and 35.62% in p-anti-EGFR transfected cells as compared with parental and empty vector transfected cells.The tumor volume of p-anti-hEGFR treated group (2 167.97± 742) mm3 was smaller [BG)W][LM][BG(][BHDWG7*2,WK42ZQ0W]than that of the control (7 413± 2 936) mm3 and pcDNA3 treated group (6 413± 2 936) mm3 (F= 9.967, P 0.01).Also,the GFAP expression was down-regulated in antisense EGFR RNA treated groups as compared with the control groups.Conclision Antisense approach targeting EGFR can exerte growth inhibitory effect on U251 glioma cells in vitro and in vivo.