Fig. 4.

DNMT3Ab represses E-cadherin expression. a Diagram of the E-cadherin gene promoter with the transcription start site (TSS) indicated. One region (−187 bp to +177 bp) spanning a CpG island with 32 CpG sites was analysed. The short black line represents the location of the fragment detected by the ChIP assay. The E-box elements near the TSS are indicated. b, c Methylation levels at the E-cadherin promoter were detected by Q-MSP assays in DNMT3Ab-transfected MKN45 and BGC-823 cells, and DNMT3Ab knockdown MKN28 cells (*P < 0.05). d Methylation status of the E-cadherin promoter in DNMT3Ab-tranfected MKN45 cells relative to that in control cells, detected by BGS assay (left). Thirty-two individual CpG sites within the CpG island (from 187 to +177 bp) were sequenced. Each row represents a single sequence; ○ indicates unmethylated CpG sites and ● indicates methylated CpG sites. The bar graphs depict the E-cadherin promoter methylation rates (right). e The binding of DNMT3Ab to the E-cadherin promoter was detected by ChIP analysis in DNMT3Ab-tranfected cells (*P < 0.05, **P < 0.01). f, g The relative expression of E-cadherin was detected in DNMT3Ab-tranfected or -knockdown cells using qPCR. β-actin was used as an internal control (**P < 0.01). h qPCR was performed to determine the E-cadherin mRNA levels in DNMT3Ab-tranfected cells after transient transfection of siRNA targeting DNMT3Aa. β-actin was used as an internal control (**P < 0.01). i The correlation between DNMT3Ab and E-cadherin expression in 24 clinical samples (R = −0.455, *P < 0.05)