Identification of the effect of DNA methylation on Ki-67 promoter activity

Objective To investigate the effect of DNA methylation on Ki-67 promoter activity.Methods Ki-67 promoter fragments were in vitro methylated using M. Hpall, M. HhaI or M. SssI methylases respectively in the presence of S-adenosyl methionine. Methylated promoter fragments were then cloned into the firefly luciferase reporter vector pGL3-Basic, these methylated promoter luciferase reporter constructs and unmethylated Ki-67 promoter constructs were used to transfect normal cell lines ( HL-7702,HUVEC) and cancer cell lines ( A549,EJ, Hela, Kert-3,SGC-7901 ). Promoter activities were determined by Dual Luciferase Reporter Assay System. Results Transient transfection of cultured cell lines with these methylated promoter reporter plasmids showed that dense methylation with M. SssI methylase abolished the promoter activity whereas methylation with HpaII and HhaI methylase resulted in partial reduction of the basal activity of the promoter compared to the unmethylated controls. The rank according to promoter activity reduction from high to low was M. SssI group,M. HhaI group and M. HpaII group respectively. Conclusion Hypermethylation of Ki-67 promoter exerted an inhibitive effect on its transcriptional activity, which was dependent on the density and loci of DNA methylation. Key words: DNA methylation;  Ki-67 gene;  Promoter;  Transcriptional activity