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Netrin-1 Induces Epithelial–Mesenchymal Transition and Promotes Hepatocellular Carcinoma Invasiveness

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Abstract

Background

Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. We elucidated the mechanism by which netrin-1 released under hypoxic stress can induce epithelial–mesenchymal transition (EMT) to promote invasion in hepatocellular carcinoma (HCC) cells.

Methods

The expression of netrin-1 and the dependent receptors UNC5H and deleted in colorectal cancer (DCC) in HCC was examined by immunohistochemistry or western blot. The HepG2 cells were cultured in 21 % O2 (normoxia) or 1 % O2 (hypoxia) for 24 h. The release of netrin-1 from hypoxic cells was detected by ELISA. Expression of E-cadherin and vimentin were examined by western blot. Inverted microscopy or confocal microscopy was used to show the cell morphology or cytoskeletal rearrangements. Cell invasion induced by hypoxia was analyzed by Transwell chamber. Cytokine IL-8 and IL-10 mRNA levels were assessed by real-time PCR.

Results

The expression of netrin-1 was increased in HCC tissue and cell lines. The dependent receptors UNC5H and DCC were decreased in most HCC cell lines. Hypoxia induced netrin-1 release in a time-dependent manner. EMT induction was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of netrin-1. Moreover, overexpression of netrin-1 resulted in EMT induction in normoxic tumor cells. Cytoskeletal rearrangements were found to occur and cell invasion was increased in cells with netrin-1 overexpression. Lastly, mRNA of IL-8 and IL-10 were also increased after recombinant human netrin-1 treatment.

Conclusion

These results suggest that in hypoxic HCC cells, netrin-1 activates downstream signaling pathways to induce EMT activation with subsequent production of multiple inflammatory mediators which in turn promotes cancer invasion.

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Acknowledgments

This study is supported by the National Natural Science Foundation of China (No. 81000928 and No. 81000159).

Conflict of interest

None.

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Authors and Affiliations

  1. Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, People’s Republic of China

    Wei Yan, Ping Han, Zhenzhen Zhou, Wei Tu, Jiazhi Liao, Peiyuan Li, Mei Liu & Dean Tian

  2. Department of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, People’s Republic of China

    Yu Fu

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  1. Wei Yan
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  2. Ping Han
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  3. Zhenzhen Zhou
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Correspondence to Dean Tian or Yu Fu.

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Yan, W., Han, P., Zhou, Z. et al. Netrin-1 Induces Epithelial–Mesenchymal Transition and Promotes Hepatocellular Carcinoma Invasiveness. Dig Dis Sci 59, 1213–1221 (2014). https://doi.org/10.1007/s10620-013-3016-z

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  • DOI: https://doi.org/10.1007/s10620-013-3016-z

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