Structural and functional analysis of human Ki-67 gene promoter

Abstract

Objective To investigate the transcriptional regulatory elements of the core promoter of Ki-67 gene. Methods The truncated fragment of Ki-67 core promoter BK235 ( -223 to + 12) was directly cloned into pGL3-Basic vector, then transfected into Hela cells, SW1990 cells, SGC-7901 cells and A549 cells. The transcriptional activity of BK235 in each carcinoma cell line was identified using the dualluciferase reporter assay system. Four internally deleted vectors of Ki-67 promoter BK235 (-223 to + 12)were prepared, whose Sp1 binding site including (- 198 to - 172), (- 170 to - 144), (-63 to -37)and (-14 to + 12) was deleted respectively, and their transcriptional activity was tested by dual-luciferase reporter assay system to locate the core regulatory element. Results The transcriptional activity of Ki-67 core promoter BK235 (-223 to + 12) in Hela cells, SW1990 cells, SGC-7901 cells and A549cells was respectively 1.2-fold, 2.0-fold, 1.7-fold and 1.4-fold of Ki-67 core promoter BK2 + (-223 to + 30). Deletion of the putative Spl binding site within BK235 resulted in the decreases of promoter activities, especially deletion of (- 170 to - 144) yielded lowest transcriptional activity, equivalent to 16. 2%,10.4%, 6.7% and 12. 2% of BK2 + (-223 to +30) promoter in Hela cells, SW1990 cells, SGC-7901cells and A549 cells, respectively. Conclusion A new core promoter BK235 ( -223 to + 12) of Ki-67gene was cloned and It had a better transcriptional performance than BK2 + (-223 to + 12). All of four Spl binding sites of Ki-67 promoter had a positive effect on transcriptional regulation function and - 170 to -144 was the core regulatory sequence of Ki-67 promoter. Key words: Ki-67 gene; Promoter; Ttranscriptional regulation