Figure 7. miR-224-SIRT3-AMPK-mTOR-HIF-1α axis formed a feedback loop in NSCLC cells. (A) The SIRT3 overexpressing model was constructed in A549 cells using SIRT3 overexpression plasmids transfection for 24 hours. The SIRT3 level was detected by RT-PCR. (B) A549 cells (normal or SIRT3 overexpression) were treated with CAF-CMmiR-224 for 24 hours. The SIRT3-AMPK-mTOR-HIF-1α expression was detected by western blot. (C) A549 cells were treated with CAF-CMmiR-224 and/or HIF-1α inhibitor PX-478 (40 μM) for 18 hours. The SIRT3-AMPK-mTOR-HIF-1α expression was examined by western blot. (D, E) EMT markers, including E-cadherin, Vimentin, and N-cadherin of NSCLC cells were determined by western blot. (F, G) The downstream molecules, including VEGFA, ANGPT1, FLT1 and TIMP1 in A549 cells were tested by RT-PCR. *** represents P<0.001. (H) The miR-224 level was examined by RT-PCR. *** represents P<0.001 vs. Vector group, &&& represents P<0.001 vs. CAF-CMmiR-224 group. N=3.