Fig 4.
IGF-1 regulated the activation of MRC5 human primary fibroblasts.
(A-C) IGF-1 induced collagen I expression of MRC5. MRC5 was treated with or without IGF-1 for 48 or 72 hours. (A) Collagen I mRNA level was measured with real-time PCR. (B and C) Collagen I protein level was determined by western blot analysis and normalized to β-actin. (D) IGF-1 enhanced migration ability of MRC5. MRC5 was treated with or without IGF-1 for 24 or 48 hours. Then MRC5 was harvested, and transwell migration assay was used to assess the 24 hours migration ability of MRC5. (E and F) IGF-1 enhanced proliferation ability of MRC5. (E) MRC5 was treated with IGF-1 for 24 hours, the proliferation of MRC5 was determined by immunofluorescent staining with anti-BrdU (green), and nuclei were stained with DAPI (blue). Original magnification, 200×(E). (F) Quantification of proliferating cells, the chart represents the percentage of BrdU positive cells among the total MRC5. All experiments were performed in triplicate. *P<0.05. **P<0.01, ***P<0.001.
Fig 7.
Human IGF-1 antibody prevented miR-130b-3p inhibitor-induced profibrotic effects on fibroblasts.
A549 or ATII was transfected with 50 nM miR-130b-3p inhibitor, 24 hours after transfection, A549 or ATII was harvested and co-cultured with starved MRC5 with or without human IGF-1 antibody using 0.4 μm co-culture system (A and C) or 8.0 μm co-culture system (B). After 48 hours, the collagen I protein level (A), migration (B), and proliferation (C) of MRC5 was determined. Original magnification, 200×(C). (D) Persistent injury of alveolar epithelial cells caused decreased expression of miR-130b-3p, which in turn failed to depress the expression of IGF-1. IGF-1, acting as a paracrine activator, regulated the activation of fibroblast though miR-130b-3p dependent mechanisms. Altogether, the downregulation of miR-130b-3p in lung may contribute to the development of lung fibrosis. *P<0.05.