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A critical role of Sp1 transcription factor in regulating the human Ki-67 gene expression

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Tumor Biology

Abstract

Ki-67 plays a crucial role in cell proliferation as well as maintenance or regulation of cell division. The mechanism governing the Ki-67 gene expression remains unknown. Thus, we cloned the core promoter of the human Ki-67 gene and further investigated its transcriptional regulation. The putative Sp1 binding sites were confirmed by electrophoretic mobility shift assay together with an anti-Sp1 antibody-mediated supershift assay. Deletion mutagenesis and firefly luciferase reporter gene assay demonstrated the essential contribution of Sp1 on transcriptional activation of the Ki-67 gene. In this study, we first confirm that there are three Sp1 binding sites in the Ki-67 core promoter. Two Sp1 sites (one at position −159 to −145 nt and the other at position −14 to +12 nt) are mainly involved in transcriptional regulation of the Ki-67 gene. Overexpression of Sp1 can enhance the Ki-67 promoter activity. However, down-regulation of Sp1 expression using siRNA-Sp1 and mithramycin effectively inhibits the Ki-67 gene transcription. Our results suggest that Sp1 is essential for basal promoter activity of the human Ki-67 gene. Inhibition of the Ki-67 transcriptional activity through abolishment of Sp1 may provide the useful prospect for gene therapy.

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Abbreviations

Sp1:

Specificity protein 1

RT-PCR:

Reverse transcription polymerase chain reaction

siRNA:

Small interfering RNA

EMSA:

Electrophoretic mobility shift assays

SV40:

Simian virus 40 promoter

HRP:

Horseradish peroxidase

TBST:

Tris-buffered saline Tween-20

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Acknowledgments

This project is supported by grants from the National Natural Science Foundation of China (nos. 30873021 and 30972976) and the Science and Technology Department of Jiangsu Province (no. BK 2010177).

Conflicts of interest

None declared.

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Authors and Affiliations

  1. Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu, 221002, People’s Republic of China

    Hui Tian, Wang Li, Fei-Fei Chen, Jie-Hui Di, Bao-Fu Zhang, Dong-Sheng Pei & Jun-Nian Zheng

  2. Department of Clinical Laboratory, Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221001, People’s Republic of China

    Guo-Wei Qian & Ping Ma

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  1. Hui Tian
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  2. Guo-Wei Qian
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Correspondence to Jun-Nian Zheng.

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Hui Tian and Guo-Wei Qian contributed equally to this work.

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Tian, H., Qian, GW., Li, W. et al. A critical role of Sp1 transcription factor in regulating the human Ki-67 gene expression. Tumor Biol. 32, 273–283 (2011). https://doi.org/10.1007/s13277-010-0119-4

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  • DOI: https://doi.org/10.1007/s13277-010-0119-4

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