Spl-binding analysis of Ki-67 promoter with nuclear extract of Hela cells

Objective To investigate the transcription factors Spl binding sites of Ki-67 promoter that functions in the Ki-67 gene transcriptional regulation. Methods Electrophoretic mobility shift assay ( EMSA) was applied to verify the binding activity between putative Spl binding sites within Ki-67 promoter and nuclear protein of Hela cells. The nuclear extracts of HeLa cells were isolated and preincubated with and without an excess of unlabeled oligonucleotide competitors Spl probes in the presence or absence of antibody. Each reaction mixture was gel and electrophoresed. Results As indicated by EMSA, the consensus oligonucleotides A2 ( -170 to -144), A3 ( -63 to -37) , A4 ( - 14 to + 12) that contained the SP1 binding sites could bind to the nuclear extract of Hela cells. The binding could be competed by the 100 molar excess unlabeled consensus oligonucleotides competitor, but it could not be competed by the 100 molar excess unlabeled mute oligonucleotides competitor. The supershift bind could be observed when Spl antibody was added to the binding reaction. The consensus oligonucleotides Al and control oligonucleotides C could not bind to the nuclear extract of Hela cells. Conclusion These regions including - 170 to - 144, -63 to -37 and - 14 to +12 contained the Spl binding sites in the Ki-67 core promoter that could bind to the nuclear extracts of Hela cells specifically. Key words: Tumour cell;  Ki-67;  Transcription factors Spl