Single small-interfering RNA expression vector for silencing multiple transforming growth factor-β pathway components

• Published in: Nucleic acids research Vol. 33; no. 15; p. e131
• Main Authors: Jazag, Amarsanaa, Kanai, Fumihiko, Ijichi, Hideaki, Tateishi, Keisuke, Ikenoue, Tsuneo, Tanaka, Yasuo, Ohta, Miki, Imamura, Jun, Guleng, Bayasi, Asaoka, Yoshinari, Kawabe, Takao, Miyagishi, Makoto, Taira, Kazunari, Omata, Masao
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2005; Oxford Publishing Limited (England)
• Subjects: Cell Line; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - genetics; Gene Expression; Genetic Vectors; Humans; Methods Online; Phenotype; Protein-Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta - antagonists & inhibitors; Receptors, Transforming Growth Factor beta - genetics; RNA Interference; RNA, Small Interfering - biosynthesis; RNA, Small Interfering - genetics; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators - antagonists & inhibitors; Trans-Activators - genetics; Transforming Growth Factor beta - antagonists & inhibitors
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gni130

Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor β (TGF-β) pathway-related Smads—Smad2, Smad3 and Smad4—at the cellular level. We observed distinct phenotypic changes in TGF-β-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.