Fig. 6

Verification of circNR3C2 in breast cancer. a Schematic diagram showing: the genomic loci of circNR3C2; the generation of circNR3C2 through back-splicing; the back-splice site confirmed by Sanger sequencing. b The convergent and divergent primers designed for detecting linear NR3C2 transcript and circNR3C2 respectively. c RT-PCR in cDNA and gDNA with the convergent and divergent primers showing the existence of circNR3C2 in breast cancer tissue and cells. GAPDH was used as a negative control. d RT-PCR with the divergent primers in total RNA showing the expression and stability of circNR3C2 in 5 breast cancer cell lines. N = control, R = RNase R treatment. e Subcellular localization of endogenous circNR3C2 in MCF-7 cells, presented via in situ hybridization with the FAM (fluorescein amidite)-labeled oligonucleotides probes. DAPI was used for staining the nucleus. Scale bar = 100 μm. f RT-qPCR showing the expression of circNR3C2 in luminal subtype and TNBC tissues. Luminal N = 30, TNBC N = 30. g Analysis of lymph node metastasis in 60 breast cancer samples with high (N = 27) or low (N = 33) circNR3C2 expression. The median expression value was used as cutoff. h Kaplan-Meier estimate showing the impact of circNR3C2 expression on the relapse-free survival of breast cancer patients (N = 60). The median expression value of circNR3C2 was used as cutoff