Saydam Okay, Glauser Daniel L, Heid Irma, Turkeri Gulen, Hilbe Monika, Jacobs Andreas H, Ackermann Mathias, Fraefel Cornel
Institute of Virology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.
Mol Ther. 2005 Nov;12(5):803-12. doi: 10.1016/j.ymthe.2005.07.534. Epub 2005 Aug 22.
In primary glioblastomas and other tumor types, the epidermal growth factor receptor (EGFR) is frequently observed with alterations, such as amplification, structural rearrangements, or overexpression of the gene, suggesting an important role in glial tumorigenesis and progression. In this study, we investigated whether posttranscriptional gene silencing by vector-mediated RNAi to inhibit EGFR expression can reduce the growth of cultured human gli36 glioma cells. To "knock down" EGFR expression, we have created HSV-1-based amplicons that contain the RNA polymerase III-dependent H1 promoter to express double-stranded hairpin RNA directed against EGFR at two different locations (pHSVsiEGFR I and pHSVsiEGFR II). We demonstrate that both pHSVsiEGFR I and pHSVsiEGFR II mediated knock-down of transiently transfected full-length EGFR or endogenous EGFR in a dose-dependent manner. The knock-down of EGFR resulted in the growth inhibition of human glioblastoma (gli36-luc) cells both in culture and in athymic mice in vivo. Cell cycle analysis and annexin V staining revealed that siRNA-mediated suppression of EGFR induced apoptosis. Overall HSV-1 amplicons can mediate efficient and specific posttranscriptional gene silencing.
在原发性胶质母细胞瘤和其他肿瘤类型中，经常观察到表皮生长因子受体（EGFR）发生改变，如基因扩增、结构重排或过表达，这表明其在胶质肿瘤发生和进展中起重要作用。在本研究中，我们调查了通过载体介导的RNA干扰进行转录后基因沉默以抑制EGFR表达是否能降低培养的人gli36胶质瘤细胞的生长。为了“敲低”EGFR表达，我们构建了基于单纯疱疹病毒1型（HSV-1）的扩增子，其包含RNA聚合酶III依赖性H1启动子，以在两个不同位置表达针对EGFR的双链发夹RNA（pHSVsiEGFR I和pHSVsiEGFR II）。我们证明pHSVsiEGFR I和pHSVsiEGFR II均以剂量依赖性方式介导瞬时转染的全长EGFR或内源性EGFR的敲低。EGFR的敲低导致人胶质母细胞瘤（gli36-luc）细胞在体外培养和体内无胸腺小鼠中均生长受抑制。细胞周期分析和膜联蛋白V染色显示，siRNA介导的EGFR抑制诱导了细胞凋亡。总体而言，HSV-1扩增子可介导高效且特异的转录后基因沉默。
