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Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-κB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications

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Journal of Biomedical Science

Abstract

The link of proto-oncogenic protein Wnt-1 production with NF-κB 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-κB-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 subunits of NF-κB as well as Wnt-1 genes were investigated. It revealed that all the three morpholino ONs inhibited NF-κB activation in human hepatoblastoma cell line HepG2 cells along with decreased Wnt-1 production. Chromatin immunoprecipitation assay ascertained the direct binding of NF-κB-p50 to the Wnt-1 promoter. Additionally, anti-P50 and anti-P65 morpholino ONs also repressed the phosphorylation of Iκ Bα which temporarily correlated with the inhibition of NF-κB activation accompanied by decreased Wnt-1 production by HepG2 cells. In summary, NF-κB 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-κB-associated Wnt-1-producing cancers.

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Abbreviations

ChIP:

Chromatin immunoprecipitation

EMSA:

Electrophoretic mobility shift assay

Iκ Bs:

Inhibitors of NF-κB

IKK:

IκB kinase

NF-κB:

Nuclear transcription factor-κB

ONs:

Oligonucleotides

RNAi:

RNA interference

Wnt-1 :

Wingless-type MMTV integration site gene family, member 1

Wnts:

Wnt proteins

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Acknowledgements

This work was supported in part by grants from the Chi Mei Foundation CMFHT9102, CMFHT9322 and CMFHT 9401, Tainan, Taiwan.

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Authors and Affiliations

  1. Division of Hepatogastroenterology, Department of Internal Medicine, Chi Mei Medical Center, Tainan, Taiwan

    Chi-Shu Sun, Kuan-Ta Wu & Sun-Lung Tsai

  2. Division of General Surgery, Department of Surgery, Chi Mei Hospital Liouying, Tainan, Taiwan

    Hao-Hsien Lee

  3. Division of General Surgery, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan

    Yih-Huei Uen & Yu-Feng Tian

  4. Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan

    Cheng-Chen Tzeng

  5. Core Facilities for Proteomics Research and Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

    Andrew H.-J. Wang

  6. Liver Research Unit, Department of Medical Research, Chi Mei Medical Center, 901 Chung-Hwa Rd, Yung-Kang City, Tainan, 710, Taiwan

    Chia-Ju Cheng & Sun-Lung Tsai

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  1. Chi-Shu Sun
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Correspondence to Sun-Lung Tsai.

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Sun, CS., Wu, KT., Lee, HH. et al. Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-κB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications. J Biomed Sci 15, 633–643 (2008). https://doi.org/10.1007/s11373-008-9251-1

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