SBP1 promoter was hypermethylated in human cancer tissues and colon cancer cell lines, which silenced gene expression and was revered by 5-Aza-2′-Deoxycytidine (DAC).

Abstract

<p>A, SBP1 promoter was highly methylated in human colon cancer tissues. Genomic DNA from adjacent normal (N) and adenocarcinoma (T) tissue was isolated, converted and amplified for human SBP1 promoter using MS-PCR. # indicated different patients. Unmethylated (U) and methylated (M) bands were detected by DNA separation on 2% agarose gels with ethidium bromide. B, Western blotting analysis showed various expression of SBP1 in human colon cancer cell lines; HCT116 cells with stable transfection of SBP1 overexpressing plasmid was used as positive control. C, SBP1 promoter was hypermethylated in HCT116 cells. Genomic DNA from LS174T, HCT116, SW480, Caco-2 and HT-29 colon cancer cells was isolated, converted and amplified via MS-PCR for human SBP1 promoter followed by gel separation of total DNA on 2% agarose gels (U, unmethylated DNA; M, methylated DNA). D, DAC reversed SBP1 promoter methylation in HCT116 cells. DNA from HCT116 cells treated with 30 µM of DAC for 96 h was isolated and separated on 2% agarose gels after conversion and MS-PCR. E, Luciferase assay showed that DAC increased SBP1 promoter luciferase activity of HCT116 cells with transfection of a vector containing the full length promoter of SBP1 and with a treatment of 30 µM DAC or PBS for 72 hours. pGL4 vector and Renilla was co-transfected as controls (* p<0.01, compared to PBS). F, DAC restored SBP1 protein expression in HCT116 cells with DAC treatment for 72 h, analyzed by Western blotting. G. SBP1 mRNA was restored by DAC in HCT116 cells (* p<0.01, compared to PBS). Each experiment was performed at least 3 times.</p