Objective: To study the inhibitory effects of siRNA targeting epidermal growth factor receptor (EGFR) on the proliferation and invasion of human glioblastoma cells.
Method: Two siRNA expression constructs using psiRNA-NeoG2 vector, that targeted sequences of human EGFR receptor L domain (516 - 536) and catalytic domain (2400 - 2420) respectively, were constructed. Human malignant glioma cells of the line TJ905 were cultured in vitro and transfected with pcDNA3-hEGFR, anti-sense RNA, blank vector psiRNA-NeoG2 (as negative control), psiRNA-NeoG2-516, and psiRNA-NeoG2-2400 respectively mediated by LipofectAMINE. Immunofluorescence assay and Western blotting were used to detect the EGFR expression. Cell apoptosis was detected by apoptotic index (AI) using TUNEL method. Cell cycle was analyzed by flow cytometry, and cell proliferative activities were measured by MTT. The expression and enzymatic activities of matrix metalloproteinase 9 (MMP-9) were measured by Western blotting and gelatin zymography, and cell invasive capabilities were evaluated by Transwell-ECM method.
Result: Immunofluorescence assay and Western blotting showed that the expression of EGFR was down-regulated by 90% and 92% respectively in the siRNA constructs transfected groups, while down-regulated by 82% in the antisense EGFR RNA transfected cells in comparison with the TJ905 cells and the cells transfected with blank vector. TUNEL assay showed that almost no apoptotic cell was found in the parental cells or the cells transfected with blank vector, however, apoptosis was increased in antisense EGFR transfected cells (AI = 7.2) and siRNA constructs transfected cells (AI = 13.7 and 14.7; chi(2) = 31.549, P < 0.001). Flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNA constructs transfected cells than that in the parental cells, the cells transfected with blank vector, and the antisense EGFR transfected cells. MTT assay indicated that compared to the parental cells and the cells transfected with blank vector, the survival rates of transfected cells dramatically dropped down from the first day after implantation (P < 0.05), the siRNA transfected cells demonstrated much lower survival rate than the antisense EGFR transfected cells. Meanwhile, the expression and enzymatic activities of MMP-9 decreased significantly after the transfection of antisense EGFR into the TJ905 cells compared to the TJ905 cells and the cells transfected with blank vector, and were much lower in the siRNA groups than that in the antisense groups (P < 0.05). Cell invasive capability assay demonstrated the similar inhibitory results in the Transwell ECM-Matrigel study.
Conclusion: Compared with antisense approach, siRNA expression constructs targeting EGFR specifically suppresses the EGFR expression, induces gene silencing, and inhibits cell growth and invasion. The plasmid-based siRNA approach should be a new strategy in glioma gene therapy targeting EGFR.