Figure 1

miR-149 targets IL-6 in gastric stroma fibroblasts and regulates FAP expression via IL-6. (A) Schematic graph of 3′-UTR of IL-6 with the putative binding sites of miR-149. The minimum free energy (mfe) required for RNA hybridization was predicted by RNAhybrid software (mfe: −19.5 kcal/mol). (B) Effect of miR-149 mimics and miR-149 inhibitor on IL-6 expression. Luciferase activity in fibroblasts co-transfected with reporter vector with wild-type or mutant IL-6 3′-UTR and miR-149 mimics or inhibitors. (C) The miR-149 expression levels in 5 CAFs and 5 NFs established from gastric tumor tissues and matched para-tumor tissues were quantified by qRT-PCR. (D) Concentrations of IL-6 in the media of cultured CAF and NF cell lines were analyzed by ELISA. (E) The levels of miR-149 expression correlate inversely with IL-6 expression in CAFs and NFs. (F) The FAP expression levels in CAF or NF transfected with controls, miR-149 or anti-miR-149 (CAF^NC, CAF^miR-149, NF^anti-miR-149 and NF^anti-NC, respectively) as analyzed by flow cytometry. (G) The relative FAP mRNA levels in CAF^NC, CAF^miR-149, NF^anti-miR-149 and NF^anti-NC were detected by qRT-PCR. (H) Concentration of IL-6 in the media of cultured CAF^NC, CAF^miR-149, NF^anti-miR-149 and NF^anti-NC. (I, J) Relative FAP levels in CAF^miR-149and NF^anti-miR-149 in the presence or absence of IL-6 or IL-6 Ab were analyzed by flow cytometry (I) and qRT-PCR (J). All data represent means ± SD of three independent experiments. *P < 0.05, **P < 0.01, student's t-test.