[Inhibitory effects of siRNA targeting epidermal growth factor receptor on proliferation and invasion of human glioblastoma cells].
Department of Neurosurgery, Tianjin Medical University General Hospital and Lab of Neuro-oncology, Tianjin Neurological Institute, Tianjin 300052, China. Zhonghua yi xue za zhi
To study the inhibitory effects of siRNA targeting epidermal growth factor receptor (EGFR) on the proliferation and invasion of human glioblastoma cells.
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.
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.
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.
Available from: Saroj Mathupala
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ABSTRACT: RNA interference (RNAi) is a recently discovered, powerful molecular mechanism that can be harnessed to engineer gene-specific silencing in mammalian tissues. A mechanism, where short double-stranded RNA (dsRNA) molecules, when introduced into cells elicit specific "knock-down" of gene expression via degradation of targeted messenger RNA, has lately become the technique of choice for analysis of gene function in oncology research. Thus, RNAi is currently being extensively evaluated as a potential therapeutic strategy against malignant gliomas, since surgical, radiological, and chemotherapeutic interventions during the past few decades have done little to improve the poor prognosis rate for patients with these dreaded tumors. This review summarizes the pre-clinical studies that are currently underway to test the validity of RNAi as a potential therapeutic strategy against malignant gliomas, and discusses the potential technical hurdles that remain to be overcome before the technique can become a promising clinical therapy to combat this frequently lethal disease.
Available from: Gerard T Redpath
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ABSTRACT: The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.
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