Clinical specimen

Adjacent normal tissues (> 5 cm away from tumors), cancerous tissues and blood samples were gathered from 20 patients with histologically proven breast cancer after operation in Hainan General Hospital. A priori power analysis (G*Power software) was performed to estimate sample size needed to yield 80% power for detecting a significant (P < 0.05) effect of treatment [27]. Meanwhile, the data of 20 patients here conformed to normal distribution and could be used for t test. Patients did not receive any chemotherapy or radiotherapy before. Moreover, they had no major immune diseases (such as collagen vascular disease, systemic lupus erythematosus or scleroderma) and other malignant tumors. Besides, normal serum specimens were harvested from 20 healthy individuals. Venous blood samples (3 mL) were obtained from the median cubital vein of the individuals, which was added into a vacutainer without anticoagulant and kept at room temperature for 0.5 h. After blood coagulation, serum was obtained by centrifuging for 10 min at 3000 rpm. The supernatant (300 μL) in each sample was stored in – 80 °C for the next experiments [28]. After confirmation by pathological detection, fresh cancerous and normal tissues were frozen with liquid nitrogen before maintaining at – 80 °C. The clinical characteristics of breast cancer patients are summarized in Table 1. All participants signed informed consent for specimen collection. The research was approved by the Research Ethics Committee in Hainan General Hospital.

Table 1 The count of clinical characteristics in breast cancerCell culture

Human breast cancer cells (MDA-MB-231, MCF-7, BT474 and SKBR-3) and normal breast epithelial cells (MCF-10A) were purchased from American Type Culture Collection (Manassas, VA, USA). MCF-10A, SKBR-3 and MDA-MB-231 were maintained in RPMI-1640 (Thermo, Waltham, MA, USA). MCF-7 and BT474 were cultured in Dulbecco's modified Eagle medium (DMEM, Sigma, St. Louis, MO, USA). They were maintained in culture medium containing 10% fetal bovine serum (FBS, Sigma) in a humidified incubator with 5% CO2 at 37 °C. FBS supplemented into culture medium was centrifuged at 110,000g at 4 °C for 70 min to eliminate any exosomes.

Isolation of NFs and CAFs

CAFs and NFs were separated from breast cancer specimens and adjacent normal samples, respectively. After mincing by PBS, tissues were digested by collagenase/hyaluronidase (Stem Cell, Durham, NC, USA) diluted in DMEM containing 10% FBS at 37 °C for 2 h. Following centrifugation, cells were suspended in DMEM containing 10% FBS before filtration. Cells were cultured in DMEM containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified atmosphere with 5% CO2 at 37 °C. Immunofluorescence and western blot were applied to detect FAP and α-SMA levels in CAFs and NFs.

Exosome isolation

After maintaining in serum-free medium, the culture supernatants of MCF-10A and SKBR-3 cells at 70% confluence were collected. The harvested culture medium was cleared by centrifugation (10 min at 500g and then 20 min at 20,000g). Next, the supernatant was filtered through a filter (0.22-μM, Millipore, Bedford, MA, USA). Exosome pellets were collected after centrifuging for 2 h at 100,000g and then resuspended in PBS. Pierce BCA protein assay kit (Thermo) was applied for detecting protein concentration. Exosome markers CD81 (Abcam, Cambridge, MA, USA), CD63 (Abcam), CD9 (Abcam) and GM130 (Abcam) were detected by western blot.

Transmission electron microscopy (TEM)

Exosomes were diluted with PBS and the suspensions (10 μL) were added onto the TEM grid (2 mm). The exosomes were contrasted for 1 min with uranyl-oxalate solution, after maintaining for 1 min at room temperature, and then washed with sterile distilled water. Exosomes on the grid were dried for 2 min before observing under a TEM (Hitachi, Tokyo) at 100 kV.

Nanoparticle tracking analysis (NTA)

NanoSight NS300 (Malvern, Surrey, UK) was applied for analyzing exosome diameters. For obtaining optimal conditions for NTA measurements, samples were diluted in PBS according to the concentration of the particles. NTA analytical software was applied for capture and data analysis.

PKH67-labeled exosomes

Exosomes were stained using PKH67 staining kit (Sigma) on the basis of manufacture's procedures. NFs (5 × 104 cells/well) were planted in a 24-well plate. Afterward, NFs were incubated for 24 h with PKH67-labeled exosomes (20 μg/mL) and observed under a confocal microscope (Olympus, Tokyo, Japan).

Immunofluorescence staining

NFs or CAFs were planted in a 24-well plate and incubated overnight. Following corresponding administrations, NFs or CAFs were fixed in 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. After blocking in 5% bovine serum albumin, they were incubated in anti-α-SMA (SAB5700835, 1:200, Sigma) at 4 °C overnight, before incubation in secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature. NFs or CAFs were imaged using a fluorescence microscope (Olympus).

Cell transfection

Short-hairpin RNA of SNHG14 (sh-SNHG14), overexpression plasmids including Oe-EBF1, Oe-FAM171A1 and negative controls (sh-NC and Oe-NC) were purchased from Gene Pharma (Shanghai, China). Afterward, SKBR-3 cells were transfected with sh-NC or sh-SNHG14, followed by exosome extraction. NFs were transfected with Oe-NC, Oe-EBF1 or Oe-FAM171A1. Moreover, SKBR-3-Exo, Exo/sh-NC or Exo/sh-SNHG14 was added to NFs. Cell transfection was conducted using Lipofectamine 2000 (Thermo) for 48 h.

Cell viability

NFs or CAFs were planted in a 96-well plate overnight. After corresponding administrations, NFs or CAFs were incubated with CCK-8 (10 μL, Beyotime, Shanghai, China) for 4 h at 37 °C. A microplate reader (Bio-Rad, Hercules, CA, USA) was applied to gauge the absorbance at 450 nm.

Transwell assay

After collection, NFs or CAFs (1 × 105 cells/mL) were suspended in serum-free culture medium and transferred to the upper chamber of the Transwell (Corning, Bedford, MA, USA). To the lower chamber of the Transwell, 500 μL of culture medium containing 10% PBS was addded. After 48 h, cells on the upper membrane were removed, while cells that migrated to the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet. Migrated cells were counted using a fluorescent microscope (Olympus).

Dual luciferase reporter assay

Three binding sites between EBF1 and FAM171A1 promoter regions were predicted by JASPAR. Mutant (MUT)-site 1, MUT-site 2, MUT-site 3 and wild (WT) site-1 were synthesized before cloning into pGL3 vectors (Promega, Madison, WI, USA), to construct FAM171A1-MUT1, FAM171A1-MUT2, FAM171A1-MUT3 and FAM171A1-WT vectors. The vectors were co-transfected with Oe-EBF1 and Oe-NC (Gene Pharma) into cells. After 48 h, luciferase activity was determined by a dual luciferase reporter assay system (Promega).

RNA immunoprecipitation (RIP)

A Magna RNA immunoprecipitation kit (Millipore) was applied for performing RIP assay. Cells were lysed by lysis buffer. Then, the lysate was added with the resuspended magnetic beads and incubated at 4 °C overnight with anti-EBF1 (Invitrogen) or anti-IgG (Abcam). RNA was extracted from magnetic bead–protein complex after detachment with proteinase K. RT-qPCR was applied to determine the enrichment of SNHG14.

Chromatin immunoprecipitation (ChIP)

EZ-ChIP™ Chromatin immunoprecipitation (ChIP) Kit (Millipore) was used to conduct the ChIP assay. Chromatins were sonicated into DNA fragments (200–400 bp) after cells were fixed in 1% formaldehyde. Next, anti-EBF1 (Invitrogen) or anti IgG (Abcam) was added to the supernatants before incubation at 4 °C overnight. After purification, DNA fragments were subjected to RT-qPCR analysis.

RT-qPCR

Total RNA was isolated from clinical samples, breast cancer cells or NFs with Trizol reagent. The RNA was used for cDNA synthesis with SuperScript™ III First-Strand Synthesis system (Invitrogen). RT-qPCR was conducted with SYBR Premix Ex Taq II (Takara, Tokyo, Japan) on ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Relative gene expressions were evaluated with 2−ΔΔCt method. Primers applied are listed in Table 2. GAPDH was used as the reference gene.

Table 2 Primer sequences used in RT-qPCRWestern blot

Cell lysis reagent was used for extracting total protein. Proteins were separated by SDS-PAGE gel, followed by transferring onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were incubated overnight at 4 °C in primary antibodies after blocked for 1 h in 5% skim milk. Primary antibodies were: α-SMA (SAB5700835, 1:1000, Sigma), FAP (ab207178, 1:1000, Abcam), EBF1 (ab108369, 1/5000, Abcam) and FAM171A1 (ab229247, 1:1000, Abcam). Membranes were incubated in secondary antibodies for 1 h. Enhanced chemiluminescence (Millipore) was applied for protein detection.

Statistical analyses

Results are displayed as mean ± SD. Each experiment was replicated three times. Differences among multiple groups were assessed with one-way ANOVA, followed by Tukey's post hoc test. Student’s t test was used for analyzing comparisons between two groups. Correlations of SNHG14, EBF1 and FAM171A1 were analyzed with Pearson correlation coefficient. SPSS 20.0 (IBM, Armonk, NY, USA) was employed to perform statistical analyses. P < 0.05 was considered as statistically significant.