Fig. 2
Ectopic expression of miR-16 affects the CAF secretome and reduces HGF levels. a CAF154-hTERT fibroblasts (1.5 × 105 cells/well) were reverse transfected with miR-16 in 12-well plates, and medium was harvested 72 h later, clarified by centrifugation, and analyzed with a fluorescence-based multiplex assay. Overall, 91 unique factors were quantified in the conditioned medium. The concentration of each soluble factor after miR-16 transfection is expressed as a percentage compared of miR-C-transfected CM. b Concentration of HGF in CM collected 72 h after transfection of 1.5 × 105 CAF154-hTERT fibroblasts with miR-C and miR-16 (n = 3; p < 0.0001). Western blot showing the levels of HGF and cMet in CAF154-hTERT fibroblasts 72 h after transfection with miR-16 and non-targeting miR-C. Actin is shown as a loading control. c HGF levels in the CM and western blot of CAF154-hTERT fibroblasts transfected as in c with a control miRNA (miR-C inh) and miR-16 inhibitor (miR-16 inh; n = 3, p = 0.0358). d The effect of miR-16 ectopic expression was determined in CM collected from a number of primary patient-derived fibroblasts (CAF154, n = 3, p = 0.0259; CAF226, n = 2; NF221, n = 3, p = 0.0316). HGF concentration was evaluated by ELISA. e The direct targeting of miR-16 on the 3′UTR of the HGF mRNA was determined by luciferase assay performed by transfection of 293 T cells with 100 nM miRNAs together with the pMirTarget HGF 3′UTR and a Renilla-expressing plasmid. The luciferase expression was evaluated 24 h after transfection by dual-luciferase assay and normalized to Renilla expression. To test the specificity of miR-mRNA interaction, the assay was performed also with a mutated version of the pMirTarget HGF 3′UTR in which the putative binding site of miR-16 was mutagenized (Additional file 3: Figure S3; **p = 0.002; ns, non-significant). f Correlation between circulating HGF concentration and smoke exposure (pack-years), r = Spearman correlation p value (left), and circulating HGF concentration and COPD, p = Wilcoxon test p value (right) in healthy heavy smokers (n = 90)