Fig. 1

circACTN4 is verified and characterized in BC cells. a Heatmap showed 14 representative differentially upregulated circRNAs in BC tissues compared with adjacent normal tissues. The red and green represent the upregulated and downregulated circRNAs. b Schematic illustration of circACTN4 formation via the circularization from exons 2 to exon 7 in ACTN4 gene. The back-splice junction sequences were confirmed by Sanger sequencing. c The existence of circACTN4 from cDNA and gDNA in MCF-7 cell was detected by RT-PCR with the divergent and convergent primers. CircACTN4 could only be amplified by divergent primers from cDNA but not gDNA. d The localization of circACTN4 was investigated in BC cells (magnification,× 1000, Scale bar, 25um) and tissues (magnification,× 400, Scale bar, 50um) with FISH. e Nuclear-cytoplasmic fractionation assay displayed that circACTN4 was mostly distributed in the nucleus of BC cells. U6 was considered as a nuclear control and GAPDH was used as a cytoplasmic protein control. f and g The expressions of circACTN4 and ACTN4 of BC cells were analyzed by qRT-PCR and RT-PCR after treatment with actinomycin D. h The levels of circACTN4 and ACTN4 were detected after RNase R digestion at different time points by qRT-PCR. i RT-PCR was used to detect the expression of circACTN4 and ACTN4 after with RNase R treatment. j Schematic model of wild type (WT) and mutant (Mut) sequences of two putative binding sites of USF2 on ACTN4 promoter. The relative luciferase activities were detected in MCF-7 cells co-transfected with luciferase reporter plasmids containing wild type or mutant ACTN4 promoter sequence and overexpression plasmids of USF2. k ChIP-qPCR analysis showed that USF2 was enriched at the promoter region of ACTN4, and GAPDH promoter was used as negatively control. l and m The expression levels of circACTN4 and ACTN4 were determined in BC cells after USF2 up-regulation or down-regulation by qRT-PCR. GAPDH was used as the normalizing gene in the above experiments. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001