Fig. 3

CircAAGAB acts as a sponge for miR-378 h and inhibits its effect on target genes. A Nucleus-cytoplasm fractionation of RNA in MCF-7 cells under hypoxia. Distribution of circAAGAB in MCF-7 cells was detected by quantitative RT-PCR. GAPDH: cytoplasmic marker; BCAR4: nuclear marker. B Nucleus-cytoplasm fractionation of RNA in MDA-MB-231 cells under hypoxia. C RNA fluorescence in situ hybridization. Probe labelled with digoxigenin-conjugated UTP was designed to hybridize circAAGAB. DAPI: nucleus marker. Scale bar: 20 μm. D Venn diagram of the predicted miRNAs. The circAAGAB-binding miRNA candidates were predicted by ENCORI (http://starbase.sysu.edu.cn/) and miRDB (http://mirdb.org/). * Top 5 miRNAs with the highest target score of the 12 common miRNAs are shown. E Expression levels of circAAGAB in cells overexpressing circAAGAB by quantitative RT-PCR. MCF-7 and MDA-MB-231 cells were transfected with 4 μg circAAGAB plasmids (pCIRC2T7-circAAGAB). Internal control: 18S rRNA. F, G Expression levels of miRNAs in MCF-7 (F) or MDA-MB-231 (G) cells overexpressing circAAGAB. The top 5 miRNAs with highest target score from miRDB were chosen for validation by quantitative RT-PCR. Internal control: 18S rRNA. H Schematic graph of the potential binding site of circAAGAB on miR-378 h. The binding site was aligned by ENCORI. Letters in red are the mutation site. I Luciferase reporter assay. HEK293T cells were co-transfected with pMIR-REPORT-circAAGAB or its mutant plasmid, miR-378 h mimics, and Renilla plasmid for 48 h. The luciferase signal was measured by the Dual-Glo luciferase reporter assay system. The firefly signal was normalized to Renilla signal and mimic control. J Expression levels of circAAGAB and miR-378 h in MDA-MB-231 cells under hypoxia. NDRG1: positive control of hypoxia. K Expression levels of the predicted target genes of miR-378 h in cells overexpressing miR-378 h. MDA-MB-231 cells were transfected with miR-378 h mimics. RNA levels of the target genes predicted by miRDB and DIANA (http://diana.imis.athena-innovation.gr/DianaTools/index.php) were measured by quantitative RT-PCR. Internal control: GAPDH. L Expression levels of the downstream genes in MDA-MB-231 cells overexpressing circAAGAB and/or miR-378 h by quantitative RT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001