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Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-β

Oncogene volume 24, pages 662–671 (2005)Cite this article

Abstract

The transforming growth factor-β (TGF-β)-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-β-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-β and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-β. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-β 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-β. 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-β was inhibited in the S4KD cells, which might be associated with a different regulation of integrin β7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

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References

  • Akiyoshi S, Ishii M, Nemoto N, Kawabata M, Aburatani H and Miyazono K . (2001). Jpn. J. Cancer Res., 92, 257–268.

  • Bhowmick NA, Ghiassi M, Bakin A, Aakre M, Lundquist CA, Engel ME, Arteaga CL and Moses HL . (2001). Mol. Cell Biol., 12, 27–36.

  • Brummelkamp TR, Bernards R and Agami R . (2002). Science, 296, 550–553.

  • Chen G, Gharib TG, Huang C-C, Taylor JMG, Misek DE, Kardia SLR, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM and Beer DG . (2002). Mol. Cell. Proteomics, 1, 304–313.

  • Chen CR, Kang Y and Massague J . (2001). Proc. Natl. Acad. Sci. USA, 98, 992–999.

  • Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S and Gauthier JM . (1998). EMBO J., 17, 3091–3100.

  • Derynck R, Akhurst RJ and Balmain A . (2001). Nat. Genet., 29, 117–129.

  • Derynck R, Goeddel DV, Ullrich A, Gutterman JU, Williams RD, Bringman TS and Berger WH . (1983). Cancer Res., 47, 707–712.

  • Dumont N, Bakin A and Arteaga CL . (2003). J. Biol. Chem., 278, 3275–3285.

  • Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K and Tuschl T . (2001). Nature, 411, 494–498.

  • Engel ME, McDonnell MA, Law BK and Moses HL . (1999). J. Biol. Chem., 274, 37413–37420.

  • Friess H, Yamanaka Y, Buchler M, Ebert M, Beger HG, Gold LI and Korc M . (1993). Gastroenterology, 105, 1846–1856.

  • Grau AM, Zhang L, Wang W, Ruan S, Evans DB, Abbruzzese JL, Zhang W and Chiao PJ . (1997). Cancer Res., 57, 3929–3934.

  • Gygi SP, Rochon Y, Franza BR and Aebersold R . (1999). Mol. Cell. Biol., 19, 1720–1730.

  • Hahn SA, Schutte M, Hoque ATMS, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH and Kern SE . (1996). Science, 271, 350–353.

  • Hartsough MT and Mulder KM . (1995). J. Biol. Chem., 270, 7117–7124.

  • Hocevar BA, Brown TL and Howe PH . (1999). EMBO J., 18, 1345–1356.

  • Ijichi H, Ikenoue T, Kato N, Mitsuno M, Togo G, Kato J, Kanai F, Shiratori Y and Omata M . (2001). Biochem. Biophys. Res. Commun., 289, 350–357.

  • Ijichi H, Otsuka M, Kanai F, Ikenoue T, Tateishi K, Kawakami T, Arakawa Y, Shimizu K, Miyazono K, Kawabe T and Omata M . (2004). Oncogene, 23, 1043–1051.

  • Imamura T, Kanai F, Kawakami T, Amarsanaa J, Ijichi H, Hoshida Y, Tanaka Y, Ikenoue T, Tateishi K, Kawabe T, Arakawa Y, Miyagishi M, Taira K, Yokosuka O and Omata M . (2004). Biochem. Biophys. Res. Commun., 318, 289–296.

  • Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G and Linsley PS . (2003). Nat. Biotechnol., 21, 635–637.

  • Jin H and Varner J . (2004). Br. J. Cancer, 90, 561–565.

  • Lim SP, Leung E and Krissansen GW . (1998). Immunogenetics, 48, 184–195.

  • Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler W, Vogelstein B, Brattain M and Willson JKV . (1995). Science, 268, 1336–1338.

  • Miyagishi M and Taira K . (2002). Nat. Biotech., 19, 497–500.

  • Miyaki M, Iijima T, Konishi M, Sakai K, Ishii A, Yasuno M, Hishima T, Koike M, Shitara N, Iwama T, Utsunomiya J, Kuroki T and Mori T . (1999). Oncogene, 18, 3098–3103.

  • Miyazono K, ten Dijke P and Heldin CH . (2000). Adv. Immunol., 75, 115–157.

  • Morton DM and Barrack ER . (1995). Cancer Res., 55, 2596–2602.

  • Schutte M, Hruban RH, Hedrick L, Cho KR, Nadasdy GM, Weinstein CL, Bova GS, Isaacs WB, Cairns P, Nawroz H, Sidransky D, Casero Jr RA, Meltzer PS, Hahn SA and Kern SE . (1996). Cancer Res., 56, 2527–2530.

  • Togo G, Toda N, Kanai F, Kato N, Shiratori Y, Kishi K, Imazeki F, Makuuchi M and Omata M . (1996). Cancer Res., 56, 5620–5623.

  • Verrecchia F, Chu ML and Mauviel A . (2001). J. Biol.Chem., 276, 17058–17062.

  • Wang D, Kanuma T, Mizunuma H, Takama F, Ibuki Y, Wake N, Mogi A, Shitara Y and Takenoshita S . (2000). Cancer Res., 60, 4507–4512.

  • Xie L, Law BK, Aakre ME, Edgerton M, Shyr Y, Bhowmick NA and Moses HL . (2003). Breast Cancer Res., 5, R187–R198.

  • Yokota T, Sakamoto N, Enomoto N, Tanabe Y, Miyagishi M, Maekawa S, Yi L, Kurosaki M, Taira K, Watanabe M and Mizusawa H . (2003). EMBO Rep., 4, 602–608.

  • Yu L, Hebert C and Zhang YE . (2002). EMBO J., 21, 3749–3759.

  • Zavadil J, Bitzer M, Liang D, Yang YC, Massimi A, Kneitz S, Piek E and Boettinger EP . (2001). Proc. Natl. Acad. Sci. USA, 98, 6686–6691.

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Acknowledgements

We thank R Derynck for providing the plasmid and M Tsubouchi for technical assistance. This study was supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

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Authors and Affiliations

  1. Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

    Amarsanaa Jazag, Hideaki Ijichi, Fumihiko Kanai, Takaaki Imamura, Bayasi Guleng, Miki Ohta, Jun Imamura, Yasuo Tanaka, Keisuke Tateishi, Tsuneo Ikenoue, Takayuki Kawakami, Takao Kawabe & Masao Omata

  2. Clinical Research Center, University of Tokyo Hospital, Tokyo, 113-8655, Japan

    Fumihiko Kanai & Yoshihiro Arakawa

  3. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, 260-0870, Japan

    Takaaki Imamura

  4. Division of Gastroenterology, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, 160-0023, Japan

    Tsuneo Ikenoue

  5. Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, 113-8656, Japan

    Makoto Miyagishi & Kazunari Taira

  6. Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, Ibaraki, 305-8562, Japan

    Makoto Miyagishi & Kazunari Taira

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  1. Amarsanaa Jazag
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  15. Takao Kawabe
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  16. Masao Omata
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Correspondence to Fumihiko Kanai.

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Supplementary Information accompanies the paper on Oncogene website (http://www.nature.com/onc)

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Jazag, A., Ijichi, H., Kanai, F. et al. Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-β. Oncogene 24, 662–671 (2005). https://doi.org/10.1038/sj.onc.1208102

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