Fig. 5. NMRAL2P upregulates DNMT3b for DNA methylation of ACOT7 promoter to suppress ACOT7 expression. (A) The expression of ACOT7 after NMRAL2P overexpression on Western blot and qRT-PCR assays. (B) Boxplot showing the relative expression of ACOT7 in normal and GC samples. (C) The methylation of ACOT7 promotor in GC tissues and GC cells SGC7901 and normal cells GES-1 by MSP assay. (D) The methylation of ACOT7-pGL3 in SGC7901 by dual-luciferase assay. (E) GES-1 cells were treated with 5-aza-dC (0.5, 1, 2 µM) and methylated ACOT7 was reduced by MSP. (F) In GES-1 cells, the protein and mRNA levels of ACOT7 were analyzed by Western blot analysis and qRT-PCR, respectively, after 5-aza-dC (0.5, 1, 2 µM) treatment for 48 h. (G) GES-1 cells were treated with 5-aza-dC (2 µM) for 12, 24, and 48 h, and methylated ACOT7 was reduced by MSP. (H) In GES-1 cells, the protein and mRNA levels of ACOT7 were analyzed by Western blot analysis and qRT-PCR assay after 5-aza-dC treatment for 12, 24, and 48 h. (I) The relative expression of DNMT1, DNMT3a, and DNMT3b after NMRAL2P overexpression and silencing by qRT-PCR assay. (J) The protein and mRNA levels of ACOT7 were analyzed by Western blot analysis and qRT-PCR assay after DNMT3b silencing in GES-1 cells. ^*Compared with control, p<0.05, ^**Compared with control, p<0.01, ^***Compared with control, p<0.001. ACOT7, acyl-CoA thioesterase 7; GC, gastric cancer; MSP, methylation-specific PCR; U, unmethylation; M, methylation.