Establishment and validation of RNA interference using the example of apoptosis-relevant genes

Specific inhibition of gene expression is becoming an increasingly important method for experimental strategies to influence tumor growth on a molecular level. In this work, we investigated the role of individual molecules for erucylphosphocholin (ErPC) induced apoptosis using specific, synthesized small interfering RNAs (siRNAs). The human gliobastoma cell lines A172, U118MG and U373MG were used as model system; protein expression was measured with western blot analysis. As a first step, we confirmed that the RNA interference method is in fact applicable to our in vitro model system for human brain tumor cells using a simple, morphological test. In the second step, we optimzied the RNA interference method for our model cell lines by targeting the mRNA of the cytoskeleton protein Lamin A/C. The transfection parameters optimized were the cell count and the concentrations of siRNA and transfection reagent Oligogectamin. The specificity of the employed method was confirmed using a control siRNA targeting Luciferase. In the third step, we applied the RNA interference method employing our optimized parameters to the genes APAF-1, Caspase 2 and Caspase 3, which are known to participate in cell apoptosis. We were able to show that the expression of the respective proteins can be down regulated in different glioblastoma cell lines using specific siRNA. The last part of this work investigates the functional importance of Caspase 3 and APAF-1 “knock dow