Figure 3.

VEGF is a direct target of miR-101 in human cholangiocarcinoma cells. A: Putative miR-101 binding sequence in the 3′UTR of VEGF mRNA. Three nucleotides (italics) in the miR-101 binding site were mutated to obtain the mutation reporter construct. B: VEGF 3′UTR luciferase reporter activity assay. miR-101–overexpressed or control cells were transfected with either wild-type or mutant VEGF 3′UTR reporter plasmids (indicated as WT or MU, respectively, on the x axis). Twenty-four hours after transfection, the cell lysates were obtained to determine luciferase activity by using a luminometer. Renilla luciferase plasmid was used as the internal control. C: miR-101 reduces VEGF mRNA in cholangiocarcinoma cells. VEGF mRNA levels were determined by RT-qPCR in miR-101–overexpressed and miRNA-scramble control cells. The data are shown as means ± SEM from three independent experiments. D: miR-101 reduces VEGF production in cholangiocarcinoma cells. The level of VEGF in cell culture supernatant was determined by enzyme immunoassay. The data are shown as means ± SEM from three independent experiments. E: The levels of VEGF mRNA in xenograft tumor tissues as determined by RT-qPCR. The data are shown as means ± SEM from six mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; L/control, control lentivirus; L/miR101, miR-101-1 lentivirus. ^∗P < 0.05, ^∗∗P < 0.01.