Figure 5

H. pylori-induced COX-2/PGE2 activation enhances epigenetic silencing of miR-149. (A) Methylation levels in the CpG sites rich region of miR-149 are higher in CAFs. Bisulfite sequencing was performed in 10 clones for each cell line. Open circle: unmethylated CpG site; filled circle: methylated CpG site. The ratios of methylation were summarized in the right panel. (B) miR-149 mRNA levels in CAF or NF treated or untreated with PGE2 or 5-Aza were analyzed by qRT-PCR. 5-Aza derepresses the PGE2-induced miR-149 repression. (C) Schematic graph of the 3′-UTR of EP2 with the putative binding site of miR-149. The mfe predicted by RNAhybrid is −16.3 kcal/mol. (D) Relative luciferase activity in fibroblasts co-transfected with reporter vector with wild-type or mutant 3′-UTR of EP2 and miR-149 mimics, inhibitors or controls as indicated. (E) Sketch map of PGE2 activating IL-6 through EP2 by eliminating miR-149, a common inhibitory factor of IL-6 and EP2. miR-149 targets IL-6 and EP2 for suppression in NFs (left panel). Through binding to EP2, PGE2 induces the hypermethylation and suppression of miR-149 that leads to the derepression of IL-6 and EP2 (right panel). (F) The PGE2 produced by SGC-7901 and GES-1 cells with or without H. pylori infection was measured by ELISA. (G, H) COX-2 mRNA and protein levels in SGC-7901 and GES-1 cells as in F. mRNA and protein levels were analyzed by qRT-PCR and western blotting, respectively. (I) The PGE2 produced by SGC-7901 and GES-1 cells with or without H. pylori infection and in the presence or absence of NS-398 (50 μmol/L, an inhibitor of COX-2) was measured by ELISA. (J, K) COX-2 mRNA and protein levels in SGC-7901 and GES-1 cells as in I. mRNA and protein levels were analyzed by qRT-PCR and western blotting, respectively. All data are means ± SD of three independent experiments. *P < 0.05, **P < 0.01, student's t-test.