The BC tissues and adjacent non-cancer tissues (n = 66) were obtained from The Third Hospital of Mianyang. Tissue specimens were immediately stored and kept at − 80 ℃. None of the patients had received radiotherapy or chemotherapy before operation. The Ethics Committee of The Third Hospital of Mianyang approved this study.
Cell culture and transient transfection
Five BC cells (BT-549, MDA-MB-231, MDA-MB-453, and MDA-MB-468) and normal breast epithelial MCF-10A cell were bought from ATCC (Manassas, VA, USA). All cells were incubated in DMEM medium (Gibco, Carlsbad, CA, USA), containing 10% FBS and 1% penicillin/streptomycin at 37℃ in an incubator with 5% CO2.
Short hairpin RNAs targeting circCEP85 (sh-circCEP85#1, sh-circCEP85#2, sh-circCEP85#3) were constructed by Invitrogen (Carlsbad, CA, USA), miR-1193 mimic or inhibitor (miR-1193 or anti-miR-1193) and corresponding controls were all bought from RiboBio (Guangzhou, China). IGF1 overexpression plasmids (IGF1) and vectors were obtained from RiboBio. BC cells were treated with above oligonucleotides or vectors through lipofectamine 3000 reagent (Invitrogen).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs were dissociated from BC cells or samples with RNA Extraction Kit (TaKaRa, Japan), and the concentration was quantitatively analyzed by NanoDROP 2000 (Thermo Fisher). Then, RNAs were subjected to reverse transcription through PrimeScript RT reagent kit (Exiqon, Aarhus, Denmark). The SYBR Premix Ex Taq II kit (Takara) was used to perform qRT-PCR. CircCEP85, miR-1193, and IGF1 expression levels were analyzed by using 2−∆∆Ct method. Primer sequences were shown in Table 1.
RNase R assay
About 1 µg of RNA was digested with 1 U of RNase R at 37 ℃ for 15 min. Then, they were reversely transcribed into cDNAs, and circCEP85 expression and GAPDH mRNA expression were all assessed by qRT-PCR.
Cell counting kit 8 (CCK8) assay
CCK8 assay (Beyotime, Shanghai, China) was performed to assess cell viability. Transfected BC cells were seeded into 96-well plates, and incubated for 48 h. 10 µL CCK8 reagent was added into cells and incubated for 4 h. The absorbance at 450 nm was examined.
5-ethynyl-2’-deoxyuridine (EDU) assay
Transfected BC cells were plated into 96 well plates. BeyoClick™ EdU-647 kit (beyotime, shanghai, China) was used to perform EDU assay. Cells incubated with EDU buffer. Then, 4% formaldehyde was performed to fix the cells. After that, DAPI staining was conducted for 30 min in the dark, and EDU-positive rate was assessed by a fluorescence microscopy.
Annexin V-FITC Apoptosis Detection Kit (beyotime, shanghai, China) was used to detect cell apoptosis. Annexin V-FITC and PI were used to stain the transfected BC cells. The apoptosis of cells was assessed by a flow cytometer (Becton, USA).
Transwell invasion assay
Transfected BC cells suspended with serum-free medium were seeded into the Matrigel-coated transwell chambers (Bechman Coulter, Brea, CA). After incubation for 24 h, the invaded cells were fixed with 4% paraformaldehyde for 30 min, and then stained with 0.1% crystal violet for 15 min. The pictures were obtained by using a light microscope.
Tube formation assay
A 96-well plate was coated with 50 μL Matrigel (356235, Corning, Tewksbury, MA, USA). HUVECs were resuspended in 100 μL condition medium of assigned BC cells, and then reseeded (3 × 104 cells/well) onto Matrigel-coated wells. After 12 h, a microscope (Olympus, Japan) was used to observe the cells.
Sphere formation assay
Transfected BC cells were cultivated in DMEM medium containing basic fibroblast growth factor (10 ng/ml), insulin (4 ng/ml), B27 (2%), and epidermal growth factor (100 ng/ml) in the Ultra-low attachment 6-well plate (Costar, Corning) for 10 days. All above relative factors were bought from Sigma-Aldrich. The fresh medium was replaced after 2–3 days. The microscope was used to observe and photograph the sphere formation.
Western blot analysis
The proteins were isolated from collected samples and transfected cells by RIPA buffer (Sigma-Aldrich). The proteins were separated by SDS-PAGE and transferred onto the PVDF membranes (Merck, Darmstadt, Hesse, Germany), and the PVDF membranes were then incubated with the primary antibodies, including anti-IGF1 (1:1,000, ab182408, Abcam), anti-PCNA (1:1,000, #71395, CST), anti-cleaved caspase 3 (1:1,000, #9661, CST), anti-VEGFA (1:1,000, ab52917, Abcam), anti-Naong (1:1,000, ab109250, Abcam), or anti-β-actin (1:4,000, ab7817, Abcam). Then, the secondary antibody was hatched with the membrane, and ECL kit was perform to observe the protein bands.
RNA pull-down assay
Cells were transfected with biotinylated-labelled circCEP85 (circCEP85 probe) or (oligo probe). 48 h later, the lysis buffer was used to lyse the cells, and the lysate was incubated with magnetic beads for 24 h. Finally, the bound RNA was isolated, and circCEP85 enrichment was assessed by qRT-PCR.
Dual-luciferase reporter assay
CircCEP85 and IGF1 sequences contained the wild type (WT) or mutant type (MUT) miR-1193 complementary binding sites were cloned and inserted into the pmirGLO dual luciferase reporter vectors by RiboBio, and named as circCEP85-WT, circCEP85-MUT, IGF1-WT or IGF1-MUT. BC cells were transfected with these luciferase reporter vectors and miR-1193 mimic using Lipofectamine™ 3000 kit. After 24 h, the luciferase activities were detected.
RNA immunoprecipitation (RIP) assay
According to the instructions of the Magna RIP Kit (Abcam, Cambridge, UK). BC cells were lysed by RIP lysis buffers, and cell lysates were cultured with the magnetic bead with anti-Ago2 (1:500, ab186733, Abcam) or anti-IgG. The level of miR-1193 and circCEP85 was evaluated by qPCR.
Xenograft mice model
BT549 cells transfected with sh-circCEP85#1 or sh-NC were resuspended with PBS (4 × 106 cells/200 µL PBS), and then the cell suspensions were subcutaneously injected into the mice (n = 5/group). Tumor volume was recorded every week and the mice were sacrificed for tumor weight measurement after 35 days. The tumor samples were analyzed by qRT-PCR and western blot. IGF1 and Ki-67 Immunohistochemistry (IHC) staining was conducted using SP Kit (Invitrogen) with anti-IGF1 (1:500, ab263903, Abcam) and anti-Ki-67 (1:500, ab1550, Abcam). Our research was permitted by The Third Hospital of Mianyang.
All experiments at least repeated for three times. Graphpad Prism 7.0 software was used to analyze data. The differences between two groups were analyzed by Student’s t-test, and differences among three or more group were analyzed by one-way analysis of variance. P < 0.05 was considered as statistically significant.