Fig. 4

Metformin decreased YAP activity by regulating miR-381. a HBEC, A549 and 95-D cells were treated with 10 mM metformin for indicated times. Cell viability was assessed by CCK8 assay. b, c A549 cells were incubated with metformin at various concentrations for 24, 48, and 72 h (b) and indicated times at 5 mM, 10 mM and 20 mM, respectively. Cell viability was assessed by CCK8 assay. d FISH result indicated that Metformin upregulated the RNA level of MiR-381 in the A549 cells. e, f RT-PCR and qPCR results indicated that Metformin dose- and time- dependently increased the RNA level of miR-381. g Decay of miR-381 was monitored in A549, H1299 and 95-D cells cotreated with Actinomycin D and Vehicle or 10 mM Metformin. RNA levels of miR-381 were determined by qPCR. h Decay of miR-381 was monitored in A549 and 95-D cells cotreated with Actinomycin D and indicated concentrations of Metformin. RNA levels of miR-381 were determined by qPCR. i-k RT-PCR, qPCR (i), western blot (j) and immunofluorescent staining (k) assays indicated that Metformin dose-dependently decreased the expression of YAP. l Metformin time-dependently decreased the expression of YAP by the qPCR and western blot assays. m, n A549 cells were treated with Metformin or co-treated with Metformin and miR-381 inhibitors, respectively. The expressions of YAP, CTGF and Cyr61 were analyzed by western blot (m) and qPCR (n) assays. o, p A549 cells were treated with Metformin or co-treated with Metformin and miR-381 mimics, respectively. The expressions of YAP, CTGF and Cyr61 were analyzed by western blot (o) and qPCR (p) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P < 0.05, **P < 0.01 or ***P < 0.001 indicates a significant difference between the indicated groups (two-tailed, unpaired Student’s t-test or one-way analysis of variance)