Fig. 1.

Identification of circRNAs in breast cancer. a Heatmap of CIRCscore (FBPcirc/FBPlinear) in tumor (T), adjacent normal tissue (AN) and blood sample (B) from six breast cancer patients. b Expression of six circRNAs with high CIRCscore were validated by RT-qPCR assay in breast tumor and adjacent normal tissue. ** represents P < 0.01. CircRNAs IDs are according to circBase through their genomic coordinates. c The ratio of circ-to-linear of circSMARCA5 in blood sample of breast cancer patients and health volunteers. Total RNA from blood sample of breast cancer patients and health volunteers was extracted and detected by RT-qPCR. The expression level was normalized with β-actin as reference. *: P < 0.05 was considered statistically significant. d Schematic illustration showing the genomic region of circSMARCA5 derived from exons 15 and 16 of the SMARCA5 gene. Convergent (gray) and divergent (black) primers were designed to amplify the linear or back-splicing products (upper). Total RNA from MCF-7 cells with or without RNase R treatment was subjected to RT-PCR (lower) and further validated by Sanger sequencing (Right). e Northern blot using a junction-specific probe or an exon 15-16 probe showing the endogenous existence of circSMARCA5 and SMARCA5 mRNA from MCF-7 cells with or without RNase-R treatment (R+ or R-). The 7926 bp marker indicates the SMARCA5 full-length transcript transcribed in vitro. The 269 bp marker indicates exon 15 and exon 16 of SMARCA5 transcribed in vitro. f The nucleus and cytoplasm mRNA of MCF-7 were extracted, and SMARCA5 and circSMARCA5 expression levels were quantitated by RT-PCR. GAPDH and hU6 serve as internal references of the cytoplasm and nucleus, respectively. “**” indicates P < 0.01. g The nucleus and cytoplasm mRNA of MCF-7 were extracted, SMARCA5 and circSMARCA5 were examined by Northern blotting, and the SMARCA5 exon 15-16 probe was applied in this experiment. h Subcellular localization of circSMARCA5 and SMARCA5 in MCF-7 cells. The signals were examined by indirect RNA FISH and confocal microscopy. The nucleus was counterstained with DAPI. The circSMARCA5 probe was labled by biotin, while the SMARCA5 probe was labled by DIG. They were stained with red and green fluorescent secondary antibodies, respectively (I) The expression of circSMARCA5 detected by northern blot. MDA-MB-231, BT474, MCF-7, SKBR3 are breast cancer cell lines. MCF-10A are normal breast cell line. N1,N2,N3,N4,N5 are adjacent normal tissues. T1,T2,T3,T4 are breast cancer tissues. “**” indicates P < 0.01