Etiology and Pathogenesis > Epidermal Growth Factor | Treatment > Gene Therapy

Meeting Abstract

RNAi mediated effective inhibition of EGFP and EGF-R gene expression in malignant glioma

Arabel Vollmann, Hans-Peter Vornlocher, Anke Geick, Thilo Spruss, Rainer Apfel, Gerhard Giegerich, Ulrich Bogdahn

University of Regensburg, Regensburg, Germany; Ribopharma AG, Kulmbach, Germany

The use of small interfering RNAs (siRNAs) as mediators of a potent and highly specific mechanism to suppress gene expression, termed RNA interference (RNAi), has recently become a powerful tool to study and modulate gene function in mammalian cells. 
To elucidate the therapeutic potential of RNAi in human glioblastoma, we initially investigated siRNAs against the enhanced green fluorescent protein (EGFP) in a stably transfected human glioma cell line (U373-EGFP). 
In parallel, downregulation of β-actin as an endogenously expressed protein was shown in a variety of glioma cell lines (U373MG, U87MG, LN18, HTZ 243). 
After optimization of transfection conditions, we finally analyzed several siRNAs targeting the epidermal growth factor receptor (EGF-R), a gene commonly altered in human glioma. 
Transfection with siRNAs resulted in significant depletion of corresponding mRNA and protein levels, as shown by real-time quantitative PCR, Western Blot and fluorimetric analysis, respectively. 
The sequence specificity of the dsRNAs used was confirmed by use of dsRNA controls with three mismatch point mutations. 
Effective downregulation of protein expression levels could be observed at siRNA-concentrations as low as 0.05nM, and the effect was sustained for up to 12 days post transfection in case of EGFP. 
β-actin protein was reduced to 10-30% of controls depending on the cell lines tested. 
SiRNAs targeting the EGF-R differing in length and structure of the overhangs were investigated, showing significant differences in efficacy of gene silencing. 
This RNAi-based system of efficiently knocking down EGF-R expression in human glioma cells in vitro will allow future studies to evaluate siRNAs as therapeutic tools in this type of tumor.

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