Fig. 5

The methylation of upstream CpG islands represses pri-miR-144/451a expression by impairing enhancer-promoter interaction in HCC cells. a The sequence of a chromosome region encompassing the pri-miR-144/451a locus and the 5′-flanking sequences was analyzed, and the CpG islands were predicted. b A diagram showing the relative position of pri-miR-144/451a, the predicted CpG island and the E1 enhancer region. c ChIP-seq data from the Cistrome Project were used to analyze sites potentially bound by CTCF, DNase and DNMT1 on the pri-miR-144/451a locus. d ChIP assays were performed using lysates of para-tumor and HCC tissues. Different fragments of the pri-miR-144/miR-451a promoter were amplified and compared between tumor and para-tumor tissues (n = 7). e The normal hepatic cell line HL-7702 and HCC line HepG2 were treated with a DNMT1 inhibitor (5-Aza, 5 μM) or DMSO. The expression of the indicated miRNA transcripts was determined via qRT-PCR (n = 5). f HepG2 cells were treated with 5-Aza or DMSO and ChIP assays were performed by antibodies of H3K4me1, HeK4me3 and EZH2. The region of pri-miR-144/451a promoter was amplified (n = 4). g The DNA methylation levels on the predicted intronic CpG island and the promoter of pri-miR-144/451a in para-tumor and HCC tissues were evaluated via BSP (n = 24). h A 3C assay was performed to detect the interaction between the E1 region (orange arrows) and other different regulatory elements (black arrows) within the pri-miR-144/451a promoter (red arrows). Bars, means ± SEMs; *, P < 0.05; **, P < 0.01; ***, P < 0.001