Fig. 1
Characterization of hypoxia-responsive circular RNA circAAGAB in breast cancer cells under different oxygen concentrations. A Sequence of back-splicing junction using CircRNA Identifier (CIRI) [35]. B Gel electrophoresis of PCR products. Divergent primers (black arrows; ← →) and convergent primers (white arrows; → ←) for PCR. Genomic DNA (gDNA) or complementary DNA (cDNA) was used as the template to examine endogenous circAAGAB in MCF-7 and MDA-MB-231 cells. The PCR product length is 248 bp using divergent primers and 159 bp by using convergent primers. C Relative expression levels of circAAGAB by quantitative RT-PCR. Random primers or oligo dT were used for RT-PCR. N.D.: not detected. D Gel electrophoresis of PCR products from MCF-7 cells treated with RNase R under different oxygen concentrations. MCF-7 cells were treated with 3U RNase R for 20 min under normoxia (O2), hypoxia (N2), or re-oxygenation (Re-O2). The PCR product length of circular AAGAB is 248 bp and length of linear AAGAB is 254 bp. E Endogenous expression levels of circAAGAB in 5 breast cancer cell lines and normal breast epithelial MCF-10A cells by quantitative RT-PCR. F Quantitative RT-PCR analysis of circAAGAB in 5 breast cancer cell lines under normoxia and hypoxia. All of the quantitative RT-PCR results were normalized to the internal control 18S. *P < 0.05, **P < 0.01, ***P < 0.001