1. ¹Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2. ²Clinical Research Center, University of Tokyo Hospital, Tokyo 113-8655, Japan
3. ³Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-0870, Japan
4. ⁴Division of Gastroenterology, Institute for Adult Diseases, Asahi Life Foundation, Tokyo 160-0023, Japan
5. ⁵Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan
6. ⁶Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8562, Japan

Correspondence: F Kanai, E-mail: kanaif-int@h.u-tokyo.ac.jp

Received 16 January 2004; Revised 3 August 2004; Accepted 14 August 2004; Published online 13 December 2004.

Abstract

The transforming growth factor- (TGF-)-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--Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF- and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF- 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-. 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- was inhibited in the S4KD cells, which might be associated with a different regulation of integrin 7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.