ORIGINAL PAPER Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-jB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications Chi-Shu Sun Æ Kuan-Ta Wu Æ Hao-Hsien Lee Æ Yih-Huei Uen Æ Yu-Feng Tian Æ Cheng-Chen Tzeng Æ Andrew H.-J. Wang Æ Chia-Ju Cheng Æ Sun-Lung Tsai Received: 15 October 2007 / Accepted: 8 April 2008 / Published online: 7 May 2008 Ó National Science Council Taipei 2008 Abstract The link of proto-oncogenic protein Wnt-1 production with NF-jB activation has been functionally demonstrated in PC12 cells, a rat pheochromocytoma cell line of neural crest lineage, while it is not yet verified in human cells. The link can be indirectly supported in our previous report that functional proteomics identifies enhanced expression of NF-jB-associated Wnt-1 production in human hepatocellular carcinoma tissues. This study aimed to further validate this link in human cells using anti-sense strategy. The effects of sequence-specific anti-sense morpholino oligonucleotides (ONs) targeting against pre-mRNA sequences of human p50 and p65 sub- units of NF-jB as well as Wnt-1 genes were investigated. It revealed that all the three morpholino ONs inhibited NF- jB activation in human hepatoblastoma cell line HepG2 cells along with decreased Wnt-1 production. Chromatin immunoprecipitation assay ascertained the direct binding of NF-jB-p50 to the Wnt-1 promoter. Additionally, anti- P50 and anti-P65 morpholino ONs also repressed the phosphorylation of Ij Ba which temporarily correlated with the inhibition of NF-jB activation accompanied by decreased Wnt-1 production by HepG2 cells. In summary, NF-jB activation is critically involved in the production of Wnt-1 by HepG2 cells. These results may have important oncology implications in treating patients with NF-jB- associated Wnt-1-producing cancers. Keywords Anti-sense strategy Á Chromatin immunoprecipitation assay Á HepG2 cells Á Ij B Á Morpholino oligonucleotide Á NF-jB Á Wnt-1 protein Abbreviations ChIP Chromatin immunoprecipitation EMSA Electrophoretic mobility shift assay Ij Bs Inhibitors of NF-jB IKK IjB kinase NF-jB Nuclear transcription factor-jB ONs Oligonucleotides RNAi RNA interference Wnt-1 Wingless-type MMTV integration site gene family, member 1 Wnts Wnt proteins Electronic supplementary material The online version of this article (doi:10.1007/s11373-008-9251-1) contains supplementary material, which is available to authorized users. C.-S. Sun Á K.-T. Wu Á S.-L. Tsai Division of Hepatogastroenterology, Department of Internal Medicine, Chi Mei Medical Center, Tainan, Taiwan H.-H. Lee Division of General Surgery, Department of Surgery, Chi Mei Hospital Liouying, Tainan, Taiwan Y.-H. Uen Á Y.-F. Tian Division of General Surgery, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan C.-C. Tzeng Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan A. H.-J.Wang Core Facilities for Proteomics Research and Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan C.-J. Cheng Á S.-L. Tsai (&) Liver Research Unit, Department of Medical Research, Chi Mei Medical Center, 901 Chung-Hwa Rd, Yung-Kang City, Tainan 710, Taiwan e-mail: sltsai@mail.chimei.org.tw 123 J Biomed Sci (2008) 15:633–643 DOI 10.1007/s11373-008-9251-1