Fig. 4

circ_0001789 targets miR-140-3p. A Fluorescence in situ hybridization images showing the subcellular localization of circ_0001789 in AGS and HGC27 cells. Green: circ_0001789; blue: Nucleus. Scale bar, 100 μm. B ‘circBank’ and ‘circInteractome’ database searching identified hsa-miR-1246, hsa-miR-1286, hsa-miR-140-3p, hsa-miR-658 and hsa-miR-890 as potential targets of circ_0001789. C RNA pull-down assay using biotin-labeled control oligo or circ_0001789 probe. RT-qPCR analysis showed significant enrichment of miR-140-3p by the circ_0001789 probe. D Schematics of the binding site of circ_0001789 sequence to miR-140-3p, and the corresponding mutations. E Dual luciferase reporter assay in AGS and HGC27 cells using reporter containing a wild-type or mutated binding site, in the presence of miR-negative control or miR-140-3p mimic. F RNA immunoprecipitation RT-qPCR analysis using anti-argonaute 2 or IgG isotype was performed in AGS and HGC27 cells. G RT-qPCR was used to detect the miR-140-3p expression level in normal gastric mucosa samples of healthy controls (n = 50) and in GC tissues (n = 70). H Kaplan–Meier plot showing the overall survival of patients with GC exhibiting high or low expression of miR-140-3p (n = 35 in each group). I Pearson’s correlation analysis showed a negative correlation between circ_0001789 and miR-140-3p in patients with GC (r > 0.6; P < 0.01). J RT-qPCR was used to detect the expression level of miR-140-3p in GC cell lines (AGS, HGC27, MKN-45 and MKN-74) and normal gastric epithelial cells (GES-1). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. circ, circular RNA; miR, microRNA; hsa, Homo sapiens; GC, gastric cancer; RT-qPCR, reverse transcription-quantitative PCR