Figure 5.

COX-2/PGE₂ activates VEGF transcription in human cholangiocarcinoma cells. A–C: CCLP1 and HuCCT1 cells transfected with the VEGF promoter luciferase reporter (pVEGF-pro) were co-transfected with COX-2 expression plasmid [COX-2 open reading frame sequence cloned in pcDNA3.0 vector (pCOX2)] (A), treated with 10 μmol/L PGE₂, (B) or co-transfected with the COX-2 siRNA. C: Twenty-four hours later, the cell lysates were obtained to measure luciferase activities. The data are presented as means ± SEM from three independent experiments. D: miR-101–overexpressed and control cells were transfected with VEGF promoter luciferase reporter plasmid. Twenty-four hours after transfection, the cell lysates were obtained to measure luciferase activities. The data are presented as means ± SEM from three independent experiments. E: VEGF in supernatants of cells with indicated treatments. The production of VEGF was increased significantly by COX-2 overexpression or 10 μmol/L PGE₂ treatment. ^†P < 0.01 compared with vehicle or pcDNA3 control. Although miR-101–overexpression decreased VEGF production, this effect was prevented by co-treatment with PGE₂ or co-expression of COX-2. ^‡P < 0.01 compared with corresponding L/control cells and PEG₂ treatment or pCOX2 transfected cells. The data are presented as means ± SEM from triplicate experiments. F: The cells were incubated with 1 μg/mL actinomycin D overnight, followed by transfection with miR-101 mimic or scramble control. Left panel: Twenty-four hours later, VEGF mRNA in cells were determined by RT-qPCR. Right panel: VEGF concentration in culture supernatants was determined by enzyme immunoassay. The data are shown as means ± SEM from triplicate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; L/control, control lentivirus; L/miR101, miR-101-1 lentivirus. ^∗P < 0.05, ^∗∗P < 0.01.