Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components.
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 beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-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.
Jazag
Amarsanaa
A
Department of Gastroenterology, Graduate School of Medicine, University of Tokyo Tokyo 113-8655, Japan.
Kanai
Fumihiko
F
Ijichi
Hideaki
H
Tateishi
Keisuke
K
Ikenoue
Tsuneo
T
Tanaka
Yasuo
Y
Ohta
Miki
M
Imamura
Jun
J
Guleng
Bayasi
B
Asaoka
Yoshinari
Y
Kawabe
Takao
T
Miyagishi
Makoto
M
Taira
Kazunari
K
Omata
Masao
M
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*
D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II