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Fig. 4 | Genome Biology

Fig. 4

From: MICMIC: identification of DNA methylation of distal regulatory regions with causal effects on tumorigenesis

Fig. 4

Validation of causal DNA methylation events in liver cancer. a Representative results showing the negative regulation of HDAC11 by methylation of its distal DRE (cg03190578, 3817 bp from HDAC11 TSS). Box plot shows the high, middle, and low expression groups of HDAC11, plotted against the methylation of the distal DRE in each group. GSEA graphs show oncogenic signatures of HDAC11 by MRA. Correlation analysis and MRA for other genes are shown in Additional file 1: Figure S10. b Confirmation that methylation of the distal DRE is the causal event for HDAC11 regulation and cellular malignancy. qPCR results showing increased HDAC11 expression in PLC8024 cells treated with 5-AZA or dCas9-TET1, and decreased expression in cells transfected with dCas9-DNMT3A-3 L relative to controls. Cell migration assays showed that dCas9-DNMT3A-3 L targeting suppressed cell migration, while overexpression (OE) of HDAC11 increased tumor cell migration. Significance was determined by t-test and error bars represent ± SD. c qPCR results for 11 liver cancer genes after dCas9-DNMT3A-3 L/TET1 epigenetic editing with dCas9-only as control, labelled as ctr1 and ctr2. Three independent replicates were conducted for each experiment. Ten out of 11 DRE-target pairs were predicted to be negatively regulated by DRE methylation, CBFA2T3 was predicted to be positively regulated by methylation of the distal DRE (cg20283771).The qPCR results (p value < 0.01 by Student’s t-test) were consistent with the predictions (Additional file 1: Figure S10). d Summary of results for cell migration and proliferation assays for liver cancer genes, showing the causal effects of distal DRE methylation on cancer cell malignancy. See photos in Additional file 1: Figure S11. e The distal DRE region and promoter of each gene of interest were cloned into the pGL3 reporter vector and assayed for luciferase activity. The reporter constructs were also co-transfected with dCas9-DNMT3A-3 L (pro_enh + targeted_DNMT3A-3 L), resulting in decreased luciferase activity except CBFA2T3 positively correlated with its DRE methylation

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