Fig. 4

CyclinD1 binds to the Dicer promoter and increases the CpG island methylation to reduce Dicer expression in ICC and HIBEpic cells. a Listing of average DNA methylation rate of eight most differentially methylated genes between LV-siR-CyclinD1 and LV-NC, including four hypermethylated genes and four hypomethylated genes. b Graphic model of the CpG islands in the Dicer promoter. (c) ChIP analysis of CyclinD1, HP1α, H3K9me3 and SUV39H1 enrichment in the Dicer promoter region. IgG served as a negative control. Relative enrichment fold = [%(ChIP/Input)]/[%(IgG/Input)]. d BSP analysis of the CpG island methylation rate. Each row represents a single sequence, and every dot represents one CpG site. White and black dots represent unmethylated and methylated CpG islands, respectively. e Quantitative RT-PCR analysis of the relative levels of Dicer to β-actin mRNA transcripts in LV-siR-CyclinD1, LV-NC-CyclinD1 and LV-NC-CyclinD1 + 5-aza-dC groups. f Dual luciferase assays demonstrate that CyclinD1 attenuates the Dicer promoter-controlled luciferase activity in HEK293T and ICC cells (pDNA: empty plasmid vector; pGL3: empty pGL3 plasmid vector; pDNA-CyclinD1: pcDNA-CyclinD1 plasmid; Dicer-pGL3: The Dicer promoter inserted in the pGL3 plasmid). Data are expressed as individual mea values or the means ± SD of each group from three separate experiments. *P < 0.05, **p < 0.01, ***p < 0.001