Expression Of EGFRvⅢ And CXCR4in Human Glioblastoma And It’s Effect On Cell Proliferation, Invasiveness

Objective To investigate the expression of epidermal growth factor receptor variant Ⅲ (EGFRvⅢ) and CXCR4in human brain glioblastoma tissue and the effect of EGFRvⅢ and CXCR4on the cell proliferation and invasion in A172cells and the related mechanisms. Methods The expressions of EGFRvⅢ and CXCR4in brain glioblastoma tissues from60cases and their correspondence para-carcinoma normal tissues were detected by immunohistochemistry method. We established the A172cell line overexpressed EGFRvⅢ and confirmed the expression of EGFRvIII by RT-PCR and immunoblot. The effects of EGFRvⅢ overexpression on the A172cells proliferation were detected by MTT, cell number counting, colony formation and xenograft tumor in nude mice, respectively. Then we used scratch experiment and transwell invasion assay to assess the invasion ability of EGFRvⅢ in A172cells; lastly, we identified EGFRvIII key downstream genes (CXCR4) and further to clarify potential mechanism by which EGFRvIII promotes the invasion and metastasis of A172cells. We also knocked down CXCR4gene expression by using siRNA method and then evaluated the effects of EGFRvⅢ overexpression on cell proliferation and invasion in A172cells. Results The positive rate of EGFRvⅢ and CXCR4expression in brain glioblastoma tissues was higher than that in the correspondence para-carcinoma normal tissues (PEGFRvⅢ<0.01; PCXCR4<0.01). We established the A172cell line overexpressing EGFRvIII which was confirmed by RT-PCR and immunoblot. The cell proliferation ability of A172cells which EGFRvⅢ overexpression was increased compared with transfection empty vector as control. Overexpression of epidermal growth factor receptor variant Ⅲ (EGFRvⅢ) could promote A172cells proliferation detected by cell growth curve and MTT assay (P<0.05). Cell number of A172-EGFRvⅢcells is (9.1±1.1)×105, while the A172-Control cells is (3.5±0.2)×105. The number of colony formation in control and EGFRvⅢ overexpression groups was (50±10) and (140±25)(P<0.01), respectively. The volume and weight of xenograft tumor in EGFRvⅢ overexpression group were higher than that in control(P<0.01). The scratch experiment and transwell invasion assay showed that overexpression of EGFRvⅢ promotes the invasion of A172cells. Migration distances of A172-Control and A172-EGFRvⅢare (265±75)um and (454±85)um, respectively(P<0.01). In the transwell invasion assay, compared to control, overexpression of EGFRvⅢ promotes the invasion of A172cells (Control vs EGFRvIII:(8.0±3.1) vs (18.0±2.5),(P<0.05). Through RT-PCR and protein immunoblot, we found that EGFRvⅢ could obviously upregulate the expression of CXCR4; further function test showed that the inhibition of endogenous CXCR4by siRNA experiment significantly inhibited cell proliferation, cell invasion and metastasis mediated by overexpression of EGFRvⅢ (P<0.01). Cell growth assay showed that cell number of A172-EGFRvⅢ-CXCR4siRNA and A172-EGFRvⅢ-Scrambled control are (3.2±1.2)×105and (11.8±0.2)×105, respectively (P<0.05). The number of colony formation in A172-EGFRvⅢ-Scrambled control and A172-EGFRvⅢ-CXCR4siRNA group are98±23and40±10(P<0.01), respectively. The reduce of EGFRvⅢ-mediated cell proliferation was detected by cell growth curve and BrdU assay(P<0.05).The scratch experiment and transwell invasion assay showed that knock down of CXCR4reduces the EGFRvⅢ-Mediated invasion of A172cells.Ⅲ the scratch experiment, compared to control, the invasion of A172-EGFRvⅢ cells decreased after knock down of CXCR4(Control vs CXCR4siRNA group:(495±75) um vs (260±67) um,(P<0.05). In the transwell invasion assay, compared to control, knock down of CXCR4reduces the EGFRvⅢ-mediated invasion of A172cells (Control vs CXCR4siRNA group:(18.5±2.8)vs(10.1±2.6), P<0.05. Conclusion EGFRvⅢ and CXCR4are overexpressed in glioblastoma and which promote the cell proliferation and invasion in A172cells. Furthermore, these results suggest that EGFRvⅢ promotes cell proliferation and invasion via upregulation of CXCR4in A172cells.