In total, 13 pairs of breast cancer (BC) and adjacent normal breast tissues (NBT) (cohort 1), 6 breast cancer brain metastasis (BCBM) tissues (cohort 2), 20 BC and 20 BCBM patients’ plasma samples (cohort 3), and 53 BCBM patients’ primary tumor tissues (cohort 4) were collected from Liaocheng People’s Hospital (Liaocheng, China). CircBCBM1 expression at the tissue level was quantified in cohort 1 and 2, that at the plasma level was quantified in cohort 3, and Kaplan-Meier analysis of the correlation between circBCBM1 expression and BMFS was conducted with data of cohort 4. Specimens were identified by two pathologists independently. Clinical information of the enrolled patients was collected from their electronic medical records. Informed consents were obtained from all participants. The study was approved by the Ethics Committee of Liaocheng People’s Hospital.
Human brain-targeting breast carcinoma cell line 231-BR and its parental cell line MDA-MB-231 were kindly provided by Patricia S Steeg (National Cancer Institute, NIH, Bethesda, MD, USA). Breast cancer cell line BT-474 and T47D were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Vienna, Austria) with 5% CO2 at 37 °C.
RNA preparation and real-time quantitative PCR (RT-qPCR) analysis
For RNase R treatment, total RNA (2 μg) was incubated with or without RNase R (3 U/μg; Epicentre Technologies, Madison, WI, USA) in 1×RNase R reaction buffer for 20 min at 37 °C, and the resulting RNA was purified using an RNeasy MinElute cleanup Kit (Qiagen). For cellular RNA fractionation analysis, the cells’ nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). The total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA).
For circBCBM1 and BRD4 detection, the cDNAs were synthesized using PrimeScript RT Master Mix (Takara, Dalian, China) according to the manufacturer’s instructions. The RT-qPCR was performed using TG Green Premix Ex Taq II kit (Takara, Dalian, China) with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) as previously described . GAPDH served as an internal reference gene. For miR-125a detection, the cDNAs were synthesized using miRNA First Strand cDNA Synthesis kit (Stem-loop Method; Shanghai Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Then qPCR was performed using MicroRNAs qPCR Kit (SYBR Green Method; Shanghai Sangon Biotech, Shanghai, China). U6 served as an internal reference gene. All primer sequences are listed in Supplementary Table S1.
RNA fluorescence in situ hybridization (FISH)
Cy3-labelled circBCBM1 FISH mix probe was designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). FISH was conducted using Ribo Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China). Briefly, 231-BR cells (60-70% confluent) were fixed, permeated and hybridized with circBCBM1 probe at 37 °C overnight. The hybridization buffer was then gradually eluted with 4× saline-sodium citrate (SSC), 2× SSC and 1× SSC. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). 18S and U6 served as reference probes. The images were acquired on a Leica SP5 confocal microscope (Leica Micosystems, Mannheim, Germany).
Oligonucleotides, plasmids and transfection
siRNA, miRNA mimics and inhibitors were designed and synthesized by RiboBio (Guangzhou, China) or Sangon Biotech (Shanghai, China). Transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) or riboFECT CP transfection kit (RiboBio, Shanghai, China). The overexpressing and silencing vectors were constructed by HANBIO (Shanghai, China) as described previously . All constructs were verified by sequencing. Lentiviral particles carrying the above-mentioned vectors were generated in HEK293T cells. 231-BR cells were infected with lentivirus at a multiplicity of infection (MOI) of 30, and screened by puromycin.
Cell counting kit-8 (CCK8) and colony formation assays
For CCK8 assay, cells were seeded into 96-well plates and cultured overnight, followed by gene silencing or overexpression treatment. After culture, CCK8 (10 μL; Dojindo, Japan) solution was added to each well and incubation for 2 h. Absorbance at 450 nm was measured using microplate reader (BioTex, Houston, TX, USA). For colony formation assay, cells were seeded into 6-well plates with 1×103 cells/well. After 14 days of culture, cells were fixed with 4% (w/v) paraformaldehyde (PFA) and stained with 0.1% crystal violet.
Apoptosis detection assay
Apoptosis detection assay was conducted using PE Annexin V Apoptosis Detection Kit I (BD Pharmingen, Franklin Lakes, NJ, USA) according to the manufacturer’s procedure. Briefly, cells were harvested, washed and resuspended in 1 × binding buffer, and incubated with 5 μL PE Annexin V and 5 μL 7-AAD for 15 min. The apoptotic cells were assessed using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA).
Wound healing and transwell migration assays
For wound healing assay, cells were seeded into a 6-well plate and scraped using a pipette tip. Images were obtained using an inverted light microscope at the time points of 0 and 24 h. For transwell migration assay, cells were seeded into the upper chamber. After incubation, the cells were fixed with methanol and stained with crystal violet. Then, the non-migrated cells that remained at the top layer were removed using a cotton swab and migrated cells at the bottom of the chamber were observed and counted under a light microscope.
Six-week-old female BALB/c nu/nu mice were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). The subcutaneous tumor model was generated by subcutaneously injection (s.c) of 231-BR cells (5 × 106) into the right shoulder of the mouse. The breast cancer brain metastasis model was generated by injecting 231-BR cells (2 × 105 cells in 0.1 ml PBS) into the left ventricle of the mouse heart. Mice were euthanized after four weeks. Mouse brains were collected and stained with hematoxylin and eosin (H&E) as previously described for metastatic nodules count . All animal experiments were conducted in accordance with the protocols evaluated and approved by the Institutional Animal Care and Use Committee of Liaocheng People’s Hospital.
RNA immunoprecipitation (RIP) assay
RIP assay was conducted using EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Cells were lysed in 100 μl RIP lysis buffer and then diluted with 900 μl RIP immunoprecipitation buffer. The cell suspension was then mixed with magnetic beads conjugated with anti-Argonaute 2 (Ago2) or control anti-IgG antibody and rotated overnight at 4°C. The beads were collected and washed using RIP washing buffer and treated with Proteinase K at 55 °C for 30 min. RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) and analyzed by RT-qPCR.
Biotinylated RNA pull-down assay
The biotinylated RNA pull-down assay was performed as described previously . The 3ʹ-biotinylated miRNA and circRNA probes were designed and synthesized by RiboBio (Guangzhou, China) or GenePharma (Shanghai, China). To pull down circRNA by miRNA, 231-BR cells were transfected with biotinylated miR-125a, miR-1306, miR-34c, miR-26a, miR-10399, miR-661 or control miRNA. 48 h later, the cells were washed and lysed on ice for 10 min. Lysates were incubated with Dynabeads™ M-280 Streptavidin magnetic beads (Invitrogen, 11205D) at 4°C for 1.5 h. The bound RNAs were purified using TRIzol for RT-qPCR analysis. To pull down miRNA by circRNA, the biotinylated circBCBM1 probe was incubated with M-280 Streptavidin magnetic beads (Invitrogen, 11205D) at 4°C for 3 h to generate probe-coated magnetic beads. 231-BR cells were lysed and incubated with probe-coated beads at 4 °C overnight. After washing, the bound RNAs were extracted for RT-qPCR analysis.
Luciferase reporter assay
HEK293T cells were seeded in a 96-well plate and cultured for 24 h. The cells were co-transfected with a mixture of miRNA mimics (5 pmol) and luciferase reporter vectors (pSI-Check2) containing BRD4 3′-UTR sequences (0.16 μg), alongside with a negative control miRNA, to examine the miRNA binding ability. After 48 h, the luciferase activity was determined using a dual luciferase reporter assay system (Promega, Madison, WI, USA) following the manufacturer's protocol. Renilla luciferase activity was normalized to firefly luciferase activity and expressed as the percentage of the control.
Western blotting analysis
Proteins were extracted in RIPA lysis buffer (P0013B, Beyotime) and the concentration was determined using BCA Protein assay kit (P0010S, Beyotime). Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk and then incubated overnight with primary antibodies, including anti-BRD4 (ab128874, Abcam), anti-MMP9 (ab38898, Abcam), anti-Shh (2207s, CST), anti-Gli1 (2643s, CST) and anti-GAPDH (sc-32233, Santa Cruz Biotechnology). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG secondary antibody at room temperature. The blots were detected by chemiluminescence and imaged on an AlphaView analysis system (ProteinSimle, USA). The quantification of individual protein bands was assessed by densitometry using ImageJ software.
Total RNA from 231-BR cells or MDA-MB-231 cells was extracted using TRIzol reagent (Invitrogen, CA, USA). Ribosomal RNA was removed by Epicentre Ribo-zero rRNA Removal Kit (Epicentre, USA). The sequencing libraries were generated by NEBNext Ultra Directional RNA Library Prep Kit (NEB, Beverly, USA) following the manufacturer’s protocol. Briefly, RNA was fragmented and first-strand cDNA was synthesized using random hexamer primers and M-MuLV ReverseTranscriptase (RNaseH-). The second strand cDNA synthesis was performed using DNA Polymerase I and RNase H, with dUTP replacing dTTP in the reaction buffer. After adenylation at the 3’ ends of cDNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated for hybridization. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, US) and then treated by 3 μL USER Enzyme (NEB, Beverly, USA) at 37 °C for 15 min followed by 95 °C for 5 min. After PCR amplification, the products were purified and library quality was assessed on Agilent Bioanalyzer 2100 (Agilent Technologies, CA, USA). The index-coded library was clustered on cBot Cluster Generation System (Illumina Inc., San Diego, CA), and was sequenced on Illumina HiSeq 2500 platform and 125 bp paired-end reads were generated. An index of the reference genome was built and paired-end clean reads were mapped to the reference genome using HISAT2. The mapped reads of each sample were assembled by StringTie. Differential expression of replicated count data was examined using edgeR software package.
All in vitro experiments were repeated at least three times. The quantitative data were expressed as means ± standard error of the mean (SEM) and analyzed by t test or one-way ANOVA. BMFS was defined as the time from the date of surgery to the date of brain metastasis. BMFS was calculated with Kaplan-Meier estimates and analyzed with the log-rank test. All statistical analyses were performed using SPSS18 software (SPSS Inc., Chicago, USA). P < 0.05 was considered statistically significant.