Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA.
J Gene Med. 2013 Jan;15(1):42-50. doi: 10.1002/jgm.2693.
Epidermal growth factor receptor (EGFR) and β-catenin are two key mediators of cell signal transduction implicated in the pathogenesis of a variety of tumors. There is emerging evidence indicating that they are overexpressed in glioblastoma multiforme (GBM) and both play significant roles in GBM carcinogenesis. Moreover, down-regulating EGFR individually only provides limited therapeutic efficacy. Therefore, we aimed to determine the feasibility and efficacy of gene therapy of GBM using combinatorial inhibition of EGFR and β-catenin in view of the cross-talk between these two signaling pathways.
The down-regulatory effect of small interfering RNA (siRNA) targeting EGFR and β-catenin alone or in combination in human GBM cells U-87 MG was evaluated by Quantitative RT-PCR. Cell proliferation in the short- and long-term was investigated by alamar blue and clonogenic assays, respectively. An annexin-V assay was performed to detect apoptosis caused by siRNA treatment. The effect of downregulating EGFR and β-catenin on cell cycle progression, cell migration and invasive potential were also examined.
The siRNA treatment potently reduced gene expression of EGFR and β-catenin at the mRNA level. Simultaneous inhibition of EGFR and β-catenin greatly decreased GBM cell proliferation. Although no significant increase in apoptosis was demonstrated, combinatorial siRNA treatment delayed the progression of cell cycle with an increased proportion of cells arrested in the G0/1 phase. Furthermore, EGFR and β-catenin siRNA in combination significantly inhibited the migratory and invasive ability of GBM cells.
Simultaneous inhibition of EGFR and β-catenin expression could represent an effective therapy for human GBM, and warrants further study in vivo.
表皮生长因子受体（EGFR）和β-连环蛋白是两种关键的细胞信号转导介质，参与多种肿瘤的发病机制。有新的证据表明，它们在多形性胶质母细胞瘤（GBM）中过度表达，并且在 GBM 癌变中都发挥重要作用。此外，单独下调 EGFR 仅提供有限的治疗效果。因此，鉴于这两条信号通路之间的串扰，我们旨在确定使用 EGFR 和β-连环蛋白联合抑制对 GBM 进行基因治疗的可行性和疗效。
通过定量 RT-PCR 评估针对 EGFR 和β-连环蛋白的 siRNA 单独或联合在人 GBM 细胞 U-87 MG 中的下调作用。通过 alamar blue 和集落形成测定分别研究短期和长期的细胞增殖。通过 Annexin-V 测定检测 siRNA 处理引起的细胞凋亡。还检查了下调 EGFR 和β-连环蛋白对细胞周期进程、细胞迁移和侵袭潜力的影响。
siRNA 处理在 mRNA 水平上强力降低了 EGFR 和β-连环蛋白的基因表达。同时抑制 EGFR 和β-连环蛋白大大降低了 GBM 细胞的增殖。尽管没有显示出明显的凋亡增加，但联合 siRNA 处理延迟了细胞周期的进展，增加了处于 G0/1 期的细胞比例。此外，EGFR 和β-连环蛋白 siRNA 的联合显著抑制了 GBM 细胞的迁移和侵袭能力。
同时抑制 EGFR 和β-连环蛋白的表达可能代表一种有效的人类 GBM 治疗方法，值得进一步在体内研究。
