Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription
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Ki-67 as a cell proliferation marker is tightly associated with maintenance and regulation of the cell division. To understand the mechanism of Ki-67 gene expression and regulation, we first cloned 5′-flanking region and identified the Ki-67 core promoter. The deletion analysis and the dual luciferase reporter assay were used to locate the Ki-67 core promoter from −223 to +12 nt relative to the transcriptional initiation site, which is the TATA less, GC rich region comprised of several putative Sp1 binding sites. Compared with the hTERT promoter and Survivin promoter, the Ki-67 core promoter possessed higher transcription activity and more desirable tumor selectivity. In order to further demonstrate the contribution of transcription factor Sp1 on regulating the Ki-67 gene transcription, we confirmed three Sp1 binding sites from −170 to −145 nt, from −63 to −38 nt, and from −14 to +12 nt existed in the Ki-67 core promoter by the supershift assay. The deletion mutagenesis, together with the dual luciferase reporter assay, indicated that these Sp1 binding sites, particularly the region from −170 to −145 nt, were involved in positive regulation of the Ki-67 gene expression. Collectively, it was demonstrated that the region from −223 to +12 nt could drive the transcription of the Ki-67 gene, and the Sp1 binding site is essential to transcriptional regulation of the Ki-67 gene.
KeywordsKi-67 Core promoter Sp1 Supershift assay
This project is supported by grants from the National Natural Science Foundation of China (No. 30972976 and 81071854), the Program for New Century Excellent Talents in University (NCET-08-0700), and the Science and Technology Department of Jiangsu Province (No. BK2009089, BK2009091, and BK2010177).
Conflicts of interest
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