Fig. 3

Validation of causal DNA methylation events in gastric cancer. a Representative results showing the negative regulation of WNT5B by methylation of its distal DRE (cg02935351, 22,595 bp from WNT5B TSS). Box plot shows the high, middle, and low expression groups of WNT5B, plotted against the methylation of the distal DRE in each group. MRA analysis was implemented by the gene set enrichment analysis (GSEA) method. GSEA graphs show tumor-suppressive signatures of WNT5B by MRA. Correlation analysis and MRA for other genes are shown in Additional file 1: Figure S5. b Confirmation that methylation of the distal DRE is the causal event for WNT5B regulation and cellular malignancy. qPCR results showing increased WNT5B expression in AGS 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 increased cell migration, while overexpression (OE) of WNT5B suppressed tumor cell migration. Significance was determined by t-test and error bars represent ± SD. c Bisulfite sequencing validation of increased methylation of the CpGs surrounding the dCas9-DNMT3A-3 L targeted DRE of WNT5B. In the lollipop diagram, black circles stand for methylated Cs and white circles for unmethylated Cs. Each box below corresponds to one CpG position in the genomic sequence. The colored bars summarize the methylation states of all sequences at that position with yellow for methylated Cs and blue for unmethylated Cs. d qPCR results for eight gastric 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. All DRE-target pairs tested here showed strong anti-correlation between expression and methylation, and the qPCR results showed dCas9-TET1 targeting increased gene expression, while dCas9-DNMT3A-3 L targeting inhibited gene expression (p value < 0.01, student t-test). e Summary of cell migration assay results for eight gastric cancer genes, showing the causal effects of distal DRE methylation on cancer cell malignancy. See photos in Additional file 1: Figure S9. f 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