Fig. 8: PCDHGA9 is transcriptionally silenced by DNA promoter hypermethylation in GC.

a Promoter regions of PCDHGA9 with CpG sites indicated by BSP are shown. b, c BSP revealed that amount of methylation at the PCDHGA9 promoter was much higher in GC tissues than in normal gastric mucosa tissues. d Using BSP, we estimated the levels of promoter methylation in MKN-28, SGC-7901 and GES-1 cells and found that treatment with 5-Aza significantly decreased the methylation of the GC cell lines. e Demethylation treatment suppressed the migration ability of GC cells, f restored PCDHGA9 expression, decreased nuclear β-catenin accumulation and inhibited EMT marker expression. Kaplan-Meier survival analysis (log-rank test) showed that lower expression and higher methylation of PCDHGA9 in GC tissues were correlated with poor prognosis. g, h, i Using Kaplan-Meier survival analysis with log-rank test, compared with patients with lower PCDHGA9 expression, those with higher PCDHGA9 expression had better MFS (p = 0.0162), and patients with no methylation had better MFS than those with methylation (p = 0.0469). Based on the combination of PCDHGA9 expression and methylation (positive or negative), the patients were divided into three groups. Kaplan-Meier survival analysis (log-rank test) showed that the PCDHGA9-negative and methylated groups had the poorest outcomes, whereas the PCDHGA9-positive and unmethylated groups had the best prognosis (p = 0.0004). PCDHGA9 induces GC cell EMT and metastasis in a β-catenin-dependent manner. j Methylation of PCDHGA9 and silencing of both the Wnt signalling and TGF-β Smad2/3 pathways. k Low PCDHGA9 levels and activation of both the Wnt signalling and TGF-β Smad2/3 pathways. l Upregulation of PCDHGA9 inhibits Wnt and TGF-β Smad2/3 pathway activation. The data are presented as the mean ± SEM of three independent experiments.