Sp1-siRNA and mithramycin effectively inhibit the Ki-67 gene transcription

Abstract

Objective To investigate the regulation of Ki-67 gene transcription by the inhibition of Sp1 protein with Sp1 small interfering RNAs (Sp1-siRNA) and Sp1 binding sites competitor mithramycin.Methods Sp1 -siRNA and mithramycin ( a drug known to block activity of Sp1 family members by binding to GC-rich regions) were used to down-regulate Ki-67 gene transcription in Hela cells, OS-RC-2 cells and A549 cells. Immunoblotting assay was performed to detect the change of Sp1 protein in Hela cells. Immunohistochemistry was used to detect the change of Ki-67 protein in Hela cells, OS-RC-2 cells and A549cells. The dual-luciferase reporter assay system was used to identify the difference of transcriptional activity between Ki-67 promoter plasmid pGLBK23 terated by Sp1-siRNA or mithramycin and plasmid pGLBK23.Results After treated by Sp1-siRNA, Sp1 protein in Hela cells was increased significantly. Sp1-siRNA and mithramycin significantly decreased the Ki-67 protein in Hela cells, OS-RC-2 cells and A549 cells.The transcription activities of Ki-67 promoter were reduced to 45.9%, 64. 5% and 46.0% by Sp1-siRNA in Hela cells, OS-RC-2 cells and A549 cells, respectively. The transcriptional activities of Ki-67 promoter were affected by mithramycin in a dose-dependent manner. Mithramycin( 500 nmol/L) inhibited the Ki-67gene transcription to 34. 7%, 44. 2% and 29. 8% in Hela cells, OS-RC-2 cells and A549 cells, respectively. Conclusion Inhibition of Sp1 protein by Sp1-siRNA and mithramycin obviously decrease Ki-67gene transcription. Key words: Ki-67 promoter; Transcription factors Sp1; siRNA; Mithramycin