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Hint1 Up-Regulates IκBα by Targeting the β-TrCP Subunit of SCF E3 Ligase in Human Hepatocellular Carcinoma Cells

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Abstract

Background and Aim

There is increasing evidence that histidine triad nucleotide-binding protein 1 (HINT1) is a novel tumor suppressor. In the present study, we investigated the mechanism by which HINT1 promotes the stability of inhibitor of NF-κB α (IκBα) in the cytoplasm of hepatocellular carcinoma (HCC) cells, which was observed in our previous study (Wang et al. in Int J Cancer 124:1526–1534, 2009).

Methods

We examined HINT1 and IκBα expression in HCC cell lines and determined the effect of HINT1 overexpression and knockdown on IκBα protein and mRNA expression in these cell lines. Then, ubiquitination assays were performed to investigate the effects of HINT1 expression plasmid transfection on IκBα ubiquitination. Next, the interaction between HINT1 and β-TrCP was investigated in immunoprecipitation and immunofluorescence assays.

Results

Our data showed that increased HINT1 expression in HepG2 and SMMC7702 cells markedly increased IκBα protein levels, while decreased HINT1 expression markedly decreased them. Overexpression or knockdown of HINT1 did not alter the transcription of IκBα, but HINT1 inhibited proteasomal IκBα degradation and reduced its ubiquitination levels. This inhibition might occur because HINT1 is a component of the SCFβ-TrCP E3 ligase, which is responsible for IκBα ubiquitination and degradation.

Conclusion

This study provides new evidence that HINT1 is a regulator of IκBα through SCFβ-TrCP E3 ligase. These findings help to clarify the mechanism underlying the anticancer effects of HINT1.

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Acknowledgments

The authors thank Prof. Yi Cao for generously providing the His6-ubiquitin plasmid, and Dr. Hong Chang and Dr. Chen Zhang for their valuable technical support.

Grant support

This research was supported by the National Natural Science Foundation of China (Grant Nos. 81060204 and 81360360), the Academic Leader Project of Health and Family Planning Commission of Yunnan Province (Grant No. D-201220), the Joint Fund for Yunnan Provincial Science and Technology Department-Kunming Medical University (Grant No. 2013FB149), the Innovation Team Project of Yunnan Colleges and Universities (2014) and the Innovation Team Project of Yunnan Province (Grant No. 2015HC033) to L.W.

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    Authors and Affiliations

    1. Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming City, 650101, Yunnan Province, China

      Zhitian Shi, Yang Ke & Lin Wang

    2. Department of Gastroenterological Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming City, 650101, Yunnan Province, China

      Xuesong Wu

    3. No. 1168 Chunrongxi Road, Chenggong District, Kunming City, 650500, Yunnan Province, China

      Lin Wang

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    1. Zhitian Shi
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    Correspondence to Lin Wang.

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    Zhitian Shi, Xuesong Wu, and Yang Ke contributed equally to this article.

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    Shi, Z., Wu, X., Ke, Y. et al. Hint1 Up-Regulates IκBα by Targeting the β-TrCP Subunit of SCF E3 Ligase in Human Hepatocellular Carcinoma Cells. Dig Dis Sci 61, 785–794 (2016). https://doi.org/10.1007/s10620-015-3927-y

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