Fig. 4

miR-190 inhibits CDKN1B transcription by up-regulating its promoter methylation.  a, b Indicated cells were transiently transfected with a CDKN1B promoter-driven luciferase reporter together with pRL-TK. Transfectants were seeded into 96-well plates to determine CDKN1B promoter transcriptional activity. pRL-TK was used as the internal control to normalize transfection efficiency. Bars indicate means ± SD from three replicate assays. The symbol (*) indicates a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001). c Potential CpG Islands of human CDKN1B promoter were analyzed via MethPrimer v1.1beta software. d, e The methylation status of the CDKN1B promoter in the methylation region was determined using the MS-PCR assay in the indicated cells. A primer set was used to evaluate the methylated (M) and unmethylated (U) copies of CDKN1B promoter in the methylation region. Methylated control was used as positive control (M), whereas unmethylated control was used as negative control (U). f, g Indicated cells were pre-treated with 5-aza-2′-deoxycytidine and the cells were then subjected to Western Blot to analyze p27 protein level. β-Actin was used as a protein loading control