title: Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-beta.
abstract: The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.
authors.affiliation: Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors.forename: Amarsanaa
authors.initials: A
authors.lastname: Jazag
authors.forename: Hideaki
authors.initials: H
authors.lastname: Ijichi
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Kanai
authors.forename: Takaaki
authors.initials: T
authors.lastname: Imamura
authors.forename: Bayasi
authors.initials: B
authors.lastname: Guleng
authors.forename: Miki
authors.initials: M
authors.lastname: Ohta
authors.forename: Jun
authors.initials: J
authors.lastname: Imamura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tateishi
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Ikenoue
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kawakami
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Arakawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Miyagishi
authors.forename: Kazunari
authors.initials: K
authors.lastname: Taira
authors.forename: Takao
authors.initials: T
authors.lastname: Kawabe
authors.forename: Masao
authors.initials: M
authors.lastname: Omata
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051901:Smad4 Protein; D015534:Trans-Activators / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta