Methylation of <i>miR-376c</i>.

<p>(<b>A</b>) Location of the six CpG sites (sites I–VI) upstream of <i>miR-376c</i>, in the putative promoter region of the gene. (<b>B</b>) Bisulfite sequencing analysis of these CpG sites in HIBEpiC and HuCCT1. The unmethylated levels of the six sites were expressed as percentages of methylation reference values. A mutation in the genome sequence of HuCCT1 was found at CpG site III. (<b>C</b>) Real-time PCR analysis of <i>miR-376c</i> expression levels in HuCCT1 cells treated with the DNA-demethylating agent 5-AZA-dCR and/or the HDAC inhibitor TSA. After treatment with 10 µM of 5-AZA-dCR for 3 days, HuCCT1 cells were incubated with TSA (0.1, 0.5, or 1.0 µM) for a further 24 h. Expression levels were normalized to <i>RNU6B</i>. The expression level in untreated HuCCT1 cells was defined as 1 (lane 1). Differences among treatments were tested by ANOVA followed by Tukey's test (* <i>p</i><0.05).