Patient-derived samples
The research was authorized by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Forty seven paired tumor tissues and adjacent non-cancerous tissues were collected from BC patients who underwent surgery at the First Affiliated Hospital of Zhengzhou University between April 2017 and December 2019. All participants signed informed consent and did not received radiotherapy, chemotherapy, or other anti-tumor treatment.
Cell culture
BC cells (HCC70 and MDA-MB-231) and human mammary gland epithelial cells (MCF-10A) were bought from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in mammary epithelial cell growth medium (MEGM) BulletKit (Lonza, Basel, Switzerland) (MCF-10A cells) or Roswell Park Memorial Institute-1640 medium (Sigma, St Louis, MO, USA) (HCC70 and MDA-MB-231) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1% streptomycin/penicillin (Solarbio, Beijing, China) and maintained at 37 °C in a moist atmosphere with 5% CO2.
Cell transfection
Transient transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Small interference (si) RNA against circ_IRAK3 (si-circ_IRAK3: 5′-TCTCTCTGCTTGATATACTGC-3′) and its negative control (si-NC: 5′-TTCTCCGAACGTGTCACGTAA-3′) were synthesized by Geneseed (Guangzhou, China). MiR-603 mimic (miR-603) and its negative control (miR-NC), as well as miR-603 inhibitor (anti-miR-603) and its negative control (anti-miR-NC), were purchased from GenePharma (Shanghai, China). The full-length sequence of KIF2A was cloned into empty pcDNA3.1 vector (pcDNA) (Life Technologies, Grand Island, NY, USA) for pcDNA3.1-KIF2A (KIF2A) generation. Lentiviral vectors carrying sh-circ_IRAK3 (sh-circ_IRAK3: 5′-CCGGTCTCTCTGCTTGATATACTGCCTCGAGGCAGTATATCAAGCAGAGAGATTTTTTG-3′) and its negative control (sh-NC: 5′-CCGGTTCTCCGAACGTGTCACGTAACTCGAGTTACGTGACACGTTCGGAGAATTTTTG-3′) were constructed by Geneseed. MDA-MB-231 cells stably knockdown circ_IRAK3 were constructed by infecting with lentiviral particles and selecting with puromycin (Solarbio).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA (tissue samples and cells) was extracted with the miRNeasy Mini Kit (Qiagen, San Diego, CA, USA). Nuclear and cytoplasmic RNAs were isolated using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) based on the manufacturer’s instructions. For RNase R treatment, total RNA from BC cells was digested with 4 U/μg RNase R (Geneseed) at 70 °C for 10 min. RNA was reversely transcribed with the M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) or miScripIIRT kit (Qiagen). Quantitative-PCR was executed with the SYBR Green PCR Master Mix (Vazyme, Nanjing, China). The data were analyzed by the 2−ΔΔCt method. The primers utilized in the research were as follows: β-actin (Forward: 5′-CTTCGCGGGCGACGAT-3′); Reverse: 5′-CCACATAGGAATCCTTCTGACC-3′), circ_IRAK3: (Forward: 5′-CTCGGTCATCTGTGGCAGTA-3′; Reverse: 5′-GTGCCCAGGACCAAAGTAAT-3′), IRAK3: (Forward: 5′-AGGATTTCCGCGGTTGTGT-3′; Reverse: 5′-ACTCAACACTGCTCCCAGG-3′), KIF2A: (Forward: 5′-TCGTACCTGCATGATTGCCA-3′; Reverse: 5′-CCACTACCCTGTGAGAAGGG-3′, miR-603: (Forward: 5′-CGCGCACACACTGCAATTAC-3′; Reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′, and U6 small nuclear RNA (U6): Forward: 5′-CTCGCTTCGGCAGCACA-3′; Reverse: 5′-AACGCTTCACGAATTTGCGT-3′. β-actin or U6 was used as an internal control.
Cell cycle progression analysis
Flow cytometry assay was employed to assess cell cycle progression. In short, the transfected BC cells were harvested and fixed with cold ethanol (70%, Sigma) at 4 °C overnight. After washing with phosphate buffer saline (PBS, Thermo Fisher Scientific), the cells were treated with RNase A (100 μg/mL, Sigma) and stained with propidium iodide (PI) (50 μg/mL, Sigma). The cell distribution was evaluated with a flow cytometer (BD Biosciences, San Jose, CA, USA).
Colony formation assay
BC cells (1 × 103) were placed into 12-well plates and cultured for 2 weeks after specific vectors or oligonucleotides transfection. Thereafter, the cells were washed with PBS (Thermo Fisher Scientific) and stained with crystal violet (0.1%, ChemeGen, Shanghai, China). Finally, the colonies were photographed and counted with an inverted microscope (MTX Lab Systems, Bradenton, FL, USA).
Cell proliferation analysis
BC cells (1 × 103) were transfected with specific vectors or oligonucleotides and then seeded into 96-well plates. After culturing for 1 day, 2 days, or 3 days, the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution (100 μL, 0.5 mg/mL, Sigma) for 4 h. Subsequently, the crystals were dissolved by dimethylsulfoxide (DMSO) (150 μL, Sigma). The absorbance at 570 nm was analyzed using the Microplate Reader (Bio-Rad, Hercules, CA, USA).
Migration and invasion analysis
The transwell chambers (8 μm, Costar, Cambridge, MA, USA) were employed to analyze cell migration and invasion. It should be noted that transwell chambers used for invasion assay were precoated with Matrigel (Sigma). In brief, the upper chamber was supplemented with the serum-free medium containing transfected BC cells (1 × 105 cells) and the bottom chamber was supplemented with a complete medium containing 10% FBS. 24 h later, the cells on the upper surface of the membrane were removed and the remaining cells were fixed with paraformaldehyde (4%, Solarbio) and stained with crystal violet (0.25%, ChemeGen). The average number of migrated and invaded cells in 5 random fields was counted, and these random fields were photographed with an inverted microscope (MTX Lab Systems) at 100 × magnification.
Cell apoptosis analysis
The apoptosis of transfected BC cells was analyzed by flow cytometry assay with the Annexin V-Fluorescein Isothiocyanate (FITC)/PI Apoptosis Detection kit (Solarbio). In short, transfected BC cells were collected by centrifugation (1000×g, 5 min) and then re-suspended in binding buffer. Next, the cells were stained with Annexin V-FITC and PI for 30 min in the dark. Subsequently, the apoptotic rate was assessed with the flow cytometer (BD Biosciences).
Western blotting
Total protein (tissue samples and cells) was extracted with the RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%, SDS-PAGE, Sangon Biotech, Shanghai, China) was conducted to isolate total protein. Next, the isolated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Roche) and then blocked with tris buffered saline tween (TBST) buffer containing 5% skim milk. After washing with TBST, the membranes were incubated with primary antibodies at 4 °C overnight, including anti-Cleaved PARP (#9541, 1:1000), anti-B-cell lymphoma/leukaemia-2 (Bcl-2) (#4223, 1:1000), anti-Bcl-2-associated x (Bax) (#2772, 1:1000), anti-KIF2A (ab197988, 1:200, Abcam, Cambridge, MA, USA), and anti-β-actin (#4967, 1:1000). Subsequently, the membranes were incubated with goat anti-rabbit IgG (#7077, 1:2000). Protein bands were visualized with an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). β-actin was used as a loading control. All antibodies were purchased from Cell Signaling Technology (Santa Cruz, California, USA), except for KIF2A.
Bioinformatics analysis
The binding sites between circ_IRAK3 and miR-603 were predicted with the circBank (http://www.circbank.cn/) database. The binding sites of KIF2A in miR-603 were predicted using the miRDB database (http://mirdb.org/).
Dual-luciferase reporter assay
The luciferase vectors carrying circ_IRAK3-wild type (WT), circ_IRAK3-mutant (MUT), KIF2A-WT, or KIF2A-MUT were established by the pMIR-REPORT™ reporter vector supplied by Genechem Co., Ltd. (Shanghai, China). BC cells (at logarithmic phase) were co-transfected with luciferase a reporter vector and miR-603 or miR-NC, followed by analyzing the luciferase activity using the luciferase reporter assay kit (Promega).
RNA pull-down assay
Biotinylated circ_IRAK3-WT, circ_IRAK3-MUT, KIF2A-WT, KIF2A-MUT (bio-circ_IRAK3-WT, bio-circ_IRAK3-MUT, bio-KIF2A-WT, and bio-KIF2A-MUT) and scrambled negative control probe were synthesized by RiboBio (Guangzhou, China). For RNA pull-down assay, these probes were incubated with streptavidin magnetic beads (Thermo Fisher Scientific) to generate probe-coated beads. Afterward, these probe-coated beads were respectively incubated with the supernatant of the lysate of BC cells at 4 °C overnight. The pulled-down miRs were extracted using Trizol (Thermo Fisher Scientific) and then subjected to qRT-PCR.
Xenograft assay
The protocols of xenograft assay were approved by the Animal Care Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Tumor formation experiments were executed with 10 4-week-old female BALB/c nude mice (Vital River Laboratory, Beijing, China), which were randomly divided into 2 groups (n = 5). The mice in each group were injected with MDA-MB-231 cells (about 5.0 × 106) carrying sh-circ_IRAK3 or sh-NC. These mice were fed under Specific Pathogen Free conditions and tumor volume was measured every 5 days with a caliper from day 10 [Volume = (length × width2)/2]. On the 35th day of injection, all mice were anesthetized with xylazine (10 mg/kg, ChemeGen) and then euthanized by cervical decapitation. The tumor tissue was excised for subsequent analysis after the mice died (pupil dilation and cardiac arrest).
Statistical analysis
All data analyses were conducted with the SPSS 20.0 software (SPSS, Chicago, IL, USA). Data were shown as the mean ± standard deviation. The correlation among circ_IRAK3, miR-603, or KIF2A was assessed by Pearson’s correlation analysis. The experiments in vitro were repeated at least 3 times. The differences between the two groups were determined with paired Student’s t test or independent Student’s t test. One-way analysis of variance with Tukey’s post hoc test was employed to assess the differences among 3 or more groups. P < 0.05 was defined as a significant difference.