Fig. 3

miR-4259 expression is transcriptionally regulated by FOXO3a. (A) The results of RT-qPCR analysis of pri-miR-4259, pre-miR-4259 and mature miR-4259 expression levels in PANC-1 and PANC-1/GEM cells. (B) The luciferase promoter reporter assays used a ~ 2.4-kb fragment upstream of the start codon of the miR-4259 gene (F1-F3). PANC-1 and PANC-1/GEM cells were transfected with the reporter constructs and the luciferase activity was measured after transfection. (C) Chromatin/protein lysates were extracted from PANC-1 and PANC-1/GEM cells, following immunoprecipitation with antibodies specific for FOXO3a, and determined by RT-qPCR analysis. FOXO3a binding site (BS); negative control (NC). (D) A schematic diagram showing the FOXO3a binding sequences or mutated versions of the miR-4259 promoter (upper). The luciferase activity was measured by a dual-luciferase reporter assay (bottom). FOXO3a 3 A, constitutively active FOXO3a. (E) The LDHA, FOXO3a (upper) and miR-4259 (bottom) expression in the paired PANC-1 cells (left panel) and paired MIA PaCa-2 cells (right panel) were analyzed by Western blotting and RT-qPCR, respectively. The results are presented as the means ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01,, ***P < 0.001 and n.s. not significant (two-tailed Student’s t test)