Human breast cancer tissues (n = 30) and the adjacent nontumor tissues (n = 30) were obtained from our hospital BC patients with who hospitalized at the our hospital from 2016 to 2020. The tissues were stored at liquid nitrogen. For the RNA extraction, the tissues were treated with TRIzol reagent. Experiments in this study were ratified by the Clinical Ethnic Committee of First Affiliated Hospital of Harbin Medical University. All patients have signed the informed content before the start of experiments.
Cell lines and materials
Human BC cell lines MDA-MB-231 and MDA-MB-468 were purchased from the Cell Bank of the Chinese Academy of Sciences (China), cultured in high glucose DMEM (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Sigma, USA). Cells were placed in a 37 °C incubator filled with 5% CO2. Propofol was obtained from Sigma and used at a dose of 10 µM for cellular experiments according to the previous report . ShcircNOLC1, siSTAT3, circNOLC1 overexpressing vectors (pcD-ciR-circNOLC1), STAT3 overexpressing vectors (pcDNA-STAT3), miR-365a-3p mimics and inhibitors, and the negative controls (NC) were synthesized by Geenseed (China). Cell transfection of the oligonucleotides (50 μM) were performed by using Lipofectamine 2000 reagent (Invitrogen).
BC cells were seeded in a 6-well plate after treatment, and incubated for 12 days to form visible colonies. The colonies were then dyed with 0.5% crystal violate (Beyotime, China) in methanol for 20 min in dark. The images were captured by digital camera (Olympus, Japan).
The in vitro growth of BC cells was determined by cell counting 8 (CCK-8) kit (Thermo) in accordance with manufacturer’s description. Briefly, 5000 cells were placed into each well of 96-well plates and incubated for 24, 48, 72 and 96 h, respectively. CCK-8 solution was then added into each well to hatch for another 1 h. Absorbance at 450 nm was measured by a microplate reader (Thermo).
Cell migration and invasion
BC cells (2 × 103) cells were seeded in the upper chamber of the transwell insert (Corning, USA) with serum-free medium. Complete medium was added into the lower chambers. After incubation for 48 h, cells penetrated through the upper chambers were stained with 0.5% crystal violet and photographed.
For wound healing experiment, cells were seeded and incubated to form monolayers, followed by scratching with sterile 200 μl pipette tip. The cells were then incubated in medium containing 0.1% FBS. The images of the scratches were captured at 0 and 24 h.
Proteins extracted from BC cells using a RIPA lysis buffer containing mixture of protease inhibitors were separated by SDS-PAGE electrophoresis, shifted to NC membranes, blocked with 5% non-fat milk for 2 h at room temperature. The blots were hatched with E-cadherin, Vimentin, ABCG2, C-Myc, Bim21, Sox2, STAT3, SLC7A11, β-actin antibodies (1:2000, Abcam, USA) at 4 ℃ for one night. Subsequently, the protein bands were visualized by incubation with HRP-conjugated secondary antibodies (Abcam) and the ECL agents (Sigma).
RNA extraction and quantification
Total RNAs of tissues and BC cells were extracted via TRIzol reagent (Invitrogen) and quantified. A total of 1 µg RNA was reversely transcribed to cDNA by using First Strand Reverse Transcription kit (Thermo), followed by real-time PCR analysis with SYBR Green qPCR Master Mix (Thermo). Relative gene expression was normalized to internal control GAPDH or U6 . Relative quantification of circRNA, miRNA and mRNA expression was compared to internal control and analyzed using the 2−ΔΔCT method. Primer sequences are as following:
circNOLC1: forward primer 5′-TGAGCCACCAAAGAACCAGA-3′, reverse primer 5′- AACTTTCGCTCTGGGACCTT-3′;
miR-365a-3p: forward primer 5′- TGCGGTAATGCCCCTAAAAA-3′, reverse primer 5′- TGCAAGAGCAATAAGGATT-3′;
STAT3: forward primer 5′- CAGCAGCTTGACACACGGTA-3′, reverse primer 5′- AAACACCAAAGTGGCATGTGA-3′;
β-actin: forward primer 5′-CAGAGCAAGAGAGGCATCC-3′, reverse primer 5′- CTGGGGTGTTGAAGGTC-3′;
U6: forward primer 5′-CTCGCTTCGGCAGCACA-3′, reverse primer 5′-CTCGCTTCGGCAGCACA-3′.
BC cells were trypsinized, suspended in serum-free DMEM/F12 medium (Hyclone), and planted into each well (500 cells) of an ultra-low-attachment 24-well plate (Corning). The medium contains B27 (Thermo), epidermal growth factor (EGF, Thermo), basic fibroblast growth factor (bFGF, Thermo) and methylcellulose (Thermo). Ten days later, the formed spheres were captured and counted by using a microscope (Leica, Germany).
Luciferase reporter gene assay
The bioinformatics analysis of miR-365a-3p, circNOLC1, and STAT3 was performed in ENCORI database. The wild type and mutated sequences of circNOLC1 and STAT3 were cloned into pmirGLO vectors (QiaGene, Germany) and co-transfected into BC cells along with miR-365a-3p mimics or NC. After 48-h transfection, cells were digested and homogenized. The luciferase activity was evaluated by using the dual-Luciferase reporter assay system of Promega in accordance with manufacturer’s description.
The RNA pull-down experiment with biotin-labelled circNOLC1 probe (Gene Pharma, China) was conducted to determine the interaction between circNOLC1 and miR-365a-3p using a Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo) following the manufacturer’s protocol.
In vivo study
Female Balb/c mice (4–6 weeks old) were obtained from Vital River Laboratory. The experimental protocol was authorized by First Affiliated Hospital of Harbin Medical University. MDA-MB-231 cells (5 × 105 cells per mouse) transfected with circNOLC1 overexpressing vectors or the control vectors were subcutaneously injected into right-side back. Propofol (50 mg/kg) was intraperitoneally administrated to mice every day when tumor size approached 100 mm3. Tumors were isolated, weighted, subjected to paraffin embedding and immunohistochemical (IHC) staining with Ki-67 antibody (Abcam).
The measured data were shown as mean ± S.D of three independent repeats, and analyzed with Student’s t-test or two-way-ANOVA using the SPSS software. The significance threshold (p) was 0.05.