Fig 7. MiR-503 inhibits VEGF protein release and mRNA expression of human lung fibroblasts by direct binding to the 3’ untranslated region (UTR) of VEGF mRNA.
(A-D) Primary human fetal lung fibroblasts (HFL-1 cells) cultured in monolayer were transfected with miR-503 mimic (Black bar) or control miRNA (White bar) transfection reagent, as described in Materials and Methods. 24hr after transfection, the medium was changed to DMEM containing 0.2% FCS, with or without IL-1ß and TNF-α (1 ng/ml), TGF-ß1 (1 ng/ml), or PGE2 (1 x 10−7 M). (A, B) 3 days after transfection, the culture medium was harvested and assayed for (A) VEGF or (B) IL-8 (with or without IL-1ß and TNF-α) by ELISA. (A) Vertical axis: VEGF release (pg per 105 cells per 2 days). (B) Vertical axis: IL-8 release (pg per 105 cells per 2 days). Horizontal axis: culture condition. (C) 1 day after transfection, RNA was isolated and endogenous miR-503 expression was analyzed in the presence of control miRNA or miR-503 mimic by real-time qPCR. Vertical axis: level of miR503 expression, expressed as fold of 18s-rRNA values. (D) 2 days after transfection, RNA was isolated and assayed for VEGF mRNA by real-time qPCR. Vertical axis: level of VEGF mRNA expression, expressed as fold of 18s-rRNA values. Horizontal axis: culture conditions. *p < 0.05, ***p < 0.001. (E) Diagrams showing luciferase reporter constructs containing UTRs of human VEGF-A gene and miR-503 targeting site, and seed and full sequences of miR-503. (F) HFL-1 cells cultured in monolayer were co-transfected with a VEGF 3’ UTR-LUC construct, control vector, miR-503 mimic, and control miRNA. Cell layers were harvested 48hr after transfection, and luciferase activity was analyzed by dual luciferase assay. Vertical axis: Firefly luciferase activity, normalized to Renilla Luciferase activity expressed as a relative value to control (indicated as an open bar). Horizontal axis: culture conditions. *p < 0.05. The data presented are means ± SE from 3 separate experiments.