Fig. 4: Apatinib activates ATG7 via downregulation of miR-3657.

a The flowchart shows the inclusion criteria for the downregulated miRNAs potentially targeting ATG7 from the miRNA-seq analysis. b The predicted binding sequences for miR-3657 within the human ATG7 3′-UTR by TargetScan. The mutation sequences of ATG7 Mut are shown in red. c Luciferase activity was measured in BGC-823 cells transfected with miR-3657 mimics or control miRNA and ATG7 3′UTR-Wt or ATG7 3′UTR-Mut. Luciferase activity was normalized to that in cells transfected with control miRNA. d–f qRT-PCR was performed in vivo (d) and in BGC-823 cells (e) and HGC-27 cells (f) incubated with apatinib, to detect the expression of miR-3657. g, h qRT-PCR was performed in BGC-823 cells (g) and HGC-27 cells (h), to detect the expression of ATG7 after transfection with miR-3657 mimics or inhibitors. i, j The protein level of LC3-II was detected by western blotting in BGC-823 cells (i) and HGC-27 cells (j) transfected with miR-3657 mimics or inhibitors. The LC3-II protein level was quantified by densitometry and normalized to GAPDH, to reflect the LC3-II/GAPDH expression ratio. *P < 0.05; **P < 0.01. k BGC-823 cells and HGC-27 cells were transfected with mRFP-GFP-LC3 plasmid and miR-3657 mimics or inhibitors. Confocal microscopy analysis is shown. Bar scale, 10 μm. The numbers of GFP+ or RFP+ dots per cell are presented as the means ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding control group. ^#P < 0.05 compared with the GFP group transfected with miR-3657 inhibitors.