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Decreasing Pin1 suppresses telomerase activity by NF-κB in HCT116 cells colorectal carcinoma

The Chinese-German Journal of Clinical Oncology volume 12, pages 181–187 (2013)Cite this article

Abstract

Objective

The aim of our study was to investigate the effect of Pin1 on the telomerase activity in human colorectal carcinoma HCT116 cells.

Methods

Firstly, we transfected plasmid pGenesil-1-Pin1 (p-shRNA) using liposome (Lipofectamine 2000) into colorectal cancer HCT-116 cells to down-regulate the expression of Pin1. To detect the apoptotic rate of HCT116 cells was by cytometry (FCM). The expression of Pin1 and hTERT at RNA levels in human colorectal cancer HCT116 cells were determined by RT-PCR. To evaluate the activity of telomerase was by TRAP-silver staining. The subcellular localization and accumulative level of p-NF-κB/p65 protein at the nuclear was detected by Immunofluorescence and Western blotting. The DNA-binding activity of NF-κB/p65 was detected by electrophoretic mobility shift assay (EMSA).

Results

Using liposome into colorectal cancer HCT-116 cells, and down-regulate the expression of Pin1 (0.392 ± 0.072-fold; P = 0.001), and the apoptotic rate was increased (11.40% ± 1.54%; P < 0.05). Compared with transfected p-CON cell group, in transfected p-shRNA cell group, the transcription of hTERT was lower (0.171 ± 0.060-fold; P = 0.001) by quantitative real-time RT-PCR, and the results of TRAP-silver staining analysis suggested that the telomerase activity was significantly declined (0.384 ± 0.015-fold; P < 0.05). Furthermore, it was demonstrated by Immunofluorescence that p-NF-κB/p65 had a nuclear localization, and the level of p-NF-κB/p65 protein at the nuclear was reduced with silencing the expression of Pin1 by Western blotting. Using EMSA, it was suggested that NF-κB/p65 was able to bind to hTERT promoter, and the direct interaction was declined with silencing the expression of Pin1.

Conclusion

Taken together, silencing Pin1 may suppress activity of telomerase and the expression of hTERT by inhibiting NF-κB/p65 activity and reducing the combination of NF-κB/p65 and hTERT gene promoter.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, 030001, China

    Jianwen Sun, Lijun Fan, Meining Li, Yuehong Zhang & Niuliang Cheng

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  1. Jianwen Sun
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  2. Lijun Fan
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  3. Meining Li
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  4. Yuehong Zhang
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  5. Niuliang Cheng
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Correspondence to Niuliang Cheng.

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Supported by a grant from the Natural Science Foundation of Shanxi Province (No. 2010011047-1, 2011021035-1)

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Sun, J., Fan, L., Li, M. et al. Decreasing Pin1 suppresses telomerase activity by NF-κB in HCT116 cells colorectal carcinoma. Chin. -Ger. J. Clin. Oncol. 12, 181–187 (2013). https://doi.org/10.1007/s10330-013-1137-7

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  • DOI: https://doi.org/10.1007/s10330-013-1137-7

Key words

  • colonic cancer
  • Pin1
  • hTERT
  • NF-κB