Fig. 6

CircEYA3 performs its oncogenic functions by increasing ATP production through the miR-1294/c-Myc axis. A and B. PANC-1 cells were transfected with NC, si-circ-1 and the miR-1294 inhibitor, and MiaPaCa-2 cells were transfected with NC, pLCDH-circEYA3 and the miR-1294 mimics, as indicated. qRT-PCR and western blot analysis were performed to assess the expression of c-Myc. C, D and E. The cell proliferation ability was evaluated by CCK-8 (C), colony formation (D), and EdU incorporation assays (E). Scale bars, 100 μm. The miR-1294 inhibitor significantly reversed the reduction in cell proliferation caused by circEYA3 knockdown in PANC-1 cells, while the miR-1294 mimic further enhanced the proliferation of MiaPaCa-2 cells. F. The effects of si-circ-1 and miR-1294 or pLCDH-circEYA3 and the miR-1294 mimic on the migratory and invasive capabilities of PDAC cells were evaluated by Transwell assays. Scale bars, 100 μm. G. The effects of si-circ-1 and miR-1294 or pLCDH-circEYA3 and the miR-1294 mimic on apoptosis were evaluated by Annexin V-PE/7-AAD staining. H and I. Western blot analysis was performed to determine the protein levels of c-Myc, E-cadherin, N-cadherin, Vimentin, Snail, Bax, and cleaved caspase-3 in order to evaluate the effects of si-circ-1 and miR-1294 or pLCDH-circEYA3 and the miR-1294 mimic on PANC-1 (H) or MiaPaCa-2 (I) cells, respectively. L. CircEYA3 knockdown decreased and circEYA3 overexpression increased ATP production. M. The miR-1294 mimic decreased but the miR-1294 inhibitor induced ATP production. N. The effects of si-circ-1 and the miR-1294 inhibitor or pLCDH-circEYA3 and the miR-1294 mimic on ATP production in PANC-1 and MiaPaCa-2 cells. *P < 0.05, **P < 0.01, ***P < 0.001