Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA.
RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110beta, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells.
Czauderna
Frank
F
Atugen AG, Otto Warburg Haus (Nr. 80), Robert-Roessle-Strasse 10, 13125 Berlin, Germany.
Fechtner
Melanie
M
Aygün
Hüseyin
H
Arnold
Wolfgang
W
Klippel
Anke
A
Giese
Klaus
K
Kaufmann
Jörg
J
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D009690:Nucleic Acid Conformation; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000502:physiology; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D012320:RNA Polymerase III / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014183:Transplantation, Heterologous; D025521:Tumor Suppressor Proteins / Q000235:genetics
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D019869:Phosphatidylinositol 3-Kinases; D012320:RNA Polymerase III; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human