Fig. 1

Circ-0007766 expression increased within HER2-positive breast cancer cells A: The original images of circRNA expression in different breast cancer cells were shown by circRNA microarray. B: Scatter plots showed the differential expression of circRNA in MDA-MB-231 and MCF-10A cells. C: Scatter plots showed the differential expression of circRNA in MCF-7 and MCF-10A cells. D: Scatter plots showed the differential expression of circRNA in SK-BR-3 and MCF-10A cells. E: A Venn diagram showed the differential expression of circRNAs in different breast cancer cells and normal breast cancer cells. F: Chromosomal location and loop formation of circ-0007766. G: The expression of circ-0007766 was determined by RTFQ-PCR in 6 breast cancer cell lines and MCF-10A cells. H: Circ-0007766 was detected by FISH in the subcellular location of SK-BR-3 cells. *: P<0.05, compared with the normal breast cell MCF-10A group; **: P<0.01, compared with the normal breast cell MCF-10A group;***: P<0.001 compared with the normal breast cell MCF-10A group."

Fig. 2

Circ-0007766 showed high expression within HER2 breast cancer tissues A: BaseScope displayed representative images of the expression and localization of circ-0007766 in breast cancer and para-cancerous tissue. B: Scatter plot of circ-0007766 expression in breast cancer and para-cancerous tissues. C: Scatter plot of circ-0007766 levels in HER2-positive and HER2-negative breast cancer tissues. D: Scatter plot of circ-0007766 expression in breast cancer of different clinical grades. *: P<0.05, compared with non-HER2; **: P<0.01, compared with each other; ***: P<0.001, compared with each other; NS: Without statistically significant meaning."

Tab. 1

BaseScope detection of circ-0007766 expression in tissue"

Fig. 3

Circ-0007766 promotes breast cancer cell invasion and migration in vitro A: The level of circ-0007766 in MDA-MB-231 cells was detected by RTFQ-PCR after transfection with circ-0007766 or NC plasmid. B: The number of migrating and invasive MDA-MB-231 cells was shown by transwell assay after transfection with circ-0007766 or NC plasmid. C: The expression of circ-0007766 in SK-BR-3 cells after transfection with si-circ-0007766 or si-NC plasmid. D: The number of migrating and invasive cells in SK-BR-3 cells after transfection with si-NC and si-circ-0007766 plasmid was shown by transwell assay. *: P<0.05, compared with NC; **: P<0.01, compared with si-NC; ***: P<0.001, compared with NC or si-NC."

Fig. 4

Circ-0007766 interacts with miR-1972, a tumor suppressor gene A: The schematic diagram showed the predicted circR-NA-miRNA-HER2 regulatory network. CircRNAs, miRNAs, and HER2 on the same regulatory axis were identified by the same color. B: The expression of miR-1972 in SK-BR-3 cells was evaluated after transfection with NC mimics (miR-NC) or miR-1972 mimic. C: The migration and invasion ability of SK-BR-3 cells was determined by transwell assay after transfection with miR-NC or miR-1972 mimic. D: The level of miR-1972 in MDA-MB-231 cells was determined after transfection with miR-1972 inhibitor or inhibitor NC. E: The migration and invasion ability of MDA-MB-231 cells was determined by transwell assay after transfection with miR-1972 inhibitor or inhibitor NC. F: The top of the figure described the predicted binding site between circ-0007766 and miR-1972, as well as the mutation strategy to change the binding site between circ-0007766 and miR-1972. The relative luciferase activity of miR-1972 mimics, miR-NC, circ-0007766-3'UTR-wt, or circ-0007766-3'UTR-mut co-transfected with 293T cells was shown in the figure below. G-I: The RAP assay and RTFQ-PCR assay detected the enrichment of circ-0007766 wt and circ-0007766 mut probes for circ-0007766 and miR-1972 in MDA-MB-231 cells. J-L: The RAP assay and RTFQ-PCR assay detected the enrichment of circ-0007766 wt and circ-0007766 mut probes for circ-0007766 and miR-1972 in MDA-MB-231 cells. *: P <0.05, compared with circ-0007766 mut or miR-NC; **: P <0.01, compared with circ-0007766 wt; ***: P<0.001, compared with Inhibitor-NC, miR-NC, NC inhibitor or circ-0007766 wt; NS: No significant difference. wt: Wild type; mut: Mutant."

Fig. 5

MiR-1972 binds to HER2 mRNA and reverses the regulatory effect of circ-0007766 on HER2 expression A: The top of the figure showed the predetermined binding sites of miR-1972 on HER2 mRNA and the mutation strategies for the binding sites of HER2 mRNA with miR-1972. The bottom of the figure showed the relative luciferase activity in 293T cells after co-transfection with miR-1972 mimics, miR-NC, HER2-3'UTR-mut (HER2-mut) or HER2-3'UTR-wt (HER2-wt). B: After co-transfection with miR-NC or miR-1972 mimics, RIP assay was performed in MDA-MB-231 cells, and HER2 mRNA 3'UTR was measured by RTFQ-PCR. C: RTFQ-PCR and Western blot confirmed the expression levels of HER2 mRNA and protein in MDA-MB-231 cells after co-transfection with miR-NC+NC, miR-NC+miR-1972, miR-1972+NC or their combinations, and specific inhibitors. D: RTFQ-PCR and Western blot showed the expression levels of HER2 mRNA and protein in MDA-MB-231 cells after co-transfection with vector+miR-NC, circ-0007766+miR-NC, vector+miR-1972 mimics or circ-0007766+miR-1972 mimics. E: RTFQ-PCR and Western blot showed the expression levels of HER mRNA and protein in SK-BR-3 cells after co-transfection with si-NC+inhibitor NC, si-circ-0007766+inhibitor NC, si-NC+miR-1972 inhibitor or si-circ-0007766+miR-1972 inhibitor. *: P<0.05, compared with si-NC or inhibitor NC; **: P<0.01, compared with inhibitor NC, vector, miR-NC or si-NC; ***: P<0.001, compared with miR-NC or vector; NS: No significant difference."

Fig. 6

Circ-0007766 promotes breast cancer cell migration and invasion through miR-1972 A: The migration and invasion potential of MDA-MB-231 cells transfected with the transfection vector+miR-NC, circ-0007766+miR-NC, vector+miR-1972 mimic or circ-0007766+miR-1972 mimic was evaluated using a transwell migration and invasion assay. B: The migration and invasion ability of SK-BR-3 cells after transfection with si-NC+inhibitors NC, si-circ-0007766+inhibitors NC, si-NC+miR-1972 inhibitor or si-circ-0007766+miR-1972 inhibitor was evaluated using a transwell assay. *: P<0.05, compared with si-NC; **: P<0.01, compared with vector, miR-NC, si-NC or inhibitor NC; ***: P<0.001, compared with vector, inhibitor NC or si-NC."

Fig. 7

Expression of HER2 in breast cancer tissues and correlation of circ-0007766 and miR-1972 expression in breast cancer cells A: Expression of HER2 mRNA in unpaired TCGA breast cancer/normal tissue. B: Expression of HER2 mRNA in paired TCGA-derived breast cancer tissue and normal tissue. C: Expression of HER2 protein in breast cancer and normal tissue, shown by representative immunohistochemical staining images from the Human Protein Atlas. D: Expression of miR-1972 in different breast cancer cells and normal breast cancer cell MCF10A. E: Analysis of the correlation between circ-0007766 and miR-1972 expression using data from Fig. 1C and Fig. 6D. Circ-0007766 and miR-1972 mRNA relative expression levels were converted to log2 scale. **: P<0.01, compared with MCF10A; ***: P<0.001, compared with normal or MCF10A."