Fig. 3
The FGFR-1 receptor regulates HGF secretion, and it is targeted by miR-16. a CAF154-hTERT fibroblasts (3 × 105 cells/well) were serum-starved for 48 h and then stimulated with increasing doses of FGF-2. CM was collected after 24 h and analyzed by ELISA to evaluate the levels of secreted HGF. b CAF154-hTERT fibroblasts were transfected with the non-targeting miR-C, miR-16, a control siRNA, or a siRNA targeting FGFR-1 or HGF. After 72 h, the CM was collected and, together with a non-conditioned medium, used to stimulate the A549 cells. Western blot was performed on transfected CAF154-hTERT fibroblasts and on stimulated A549 cells to detect the activated state of cMet and its total levels, the levels of FGFR-1, and the activated form of ERK1/2. Actin is shown as a loading control. c Luciferase assay performed as in Fig. 2e, by using 293 T cells transfected with the pMirTarget FGFR-1 3′UTR and a Renilla-expressing plasmid. The FGFR-1 3′UTR displays two putative miR-16 binding sites (Additional file 3: Figure S3). d Levels of HGF in CM of CAF154-hTERT fibroblasts transfected with the control miR-C and miR-16 or a control siRNA and siRNA specific for FGFR-1 or HGF. Non-conditioned medium is shown as a negative control. e CAF154-hTERT fibroblasts were treated with 10 μM FGFR-1 inhibitor SU5402 and CM collected at the indicated times to evaluate HGF concentration by ELISA. The graph is representative of two independent experiments. f Correlation between the expression of HGF and FGFR-1 in primary fibroblasts derived from lung cancer patients and expressing high levels of HGF (R2 = 0.3607, slope = 0.3797 ± 0.1054). g CAF154-hTERT fibroblasts were transduced with lentiviral particles to stably express FGFR-1 and transfected with control miR-C and miR-16. Western blot was performed 72 h after transfection to detect MEK1/2 and FGFR-1 levels and the activated form of ERK1/2. Actin is shown as a loading control. h ELISA performed to evaluate the levels of HGF in the CM of cells transfected as described in g