Patient samples and Ethical statement
The tumor tissues of the breast cancer patients came from the affiliated hospital of Weifang Medical College. During surgery, 10 pairs of matched tumor tissues and adjacent normal tissues were collected to isolate CAFs and normal fibroblasts (NFs), and washed fresh cancer and normal tissues with PBS containing 20% antibiotics immediately. Isolation of CAFs and NFs by immunomagnetic bead cell sorting (MACS). After washing the specimen, cut it into small pieces, add DMEM/F12 medium containing 0.5% collagenase IV and 1% FBS and incubate the cell suspension for 30 min. Primary medium for fibroblasts (Dulbecco's modified DMEM-F12 medium containing 10% fetal bovine serum). The primary fibroblasts cultured from two to three generations were digested, counted and resuspended in MACS buffer. Anti-human fibroblast magnetic beads were added and purified in an immunomagnetic bead cell sorter. The purified CAFs and NFs were cultured in DMEM medium containing 10% FBS, and cells from 4 to 10 passages were used for experiments. The moral approval comes from the system ethics committee of Weifang Medical University.
Breast cancer cell lines MCF-7, MDA-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Primary fibroblasts and cancer-associated fibroblasts were grown in Dulbecco modified DMEM-F12 medium containing 10% FBS. The cell lines were kept at 37℃ in 5% CO2 humidification atmosphere and logarithmic growth phase cells were used for experiment. To prepare exosome depleted FBS, the supernatant was extracted under aseptic conditions by 110,000×g supernatant centrifugation for 18 hours at 4℃. In experiments involving exosomes, exosome depleted FBS was used in cell culture.
Isolation of exosomes
After 24 hours of culture in serum-free medium, the supernatant of normal fibroblasts and cancer-associated fibroblasts culture was collected. The cells were incubated with the complete medium containing serum to a density of about 80%, discarded the supernatant, and continued culture with serum-free medium. More than 10ml of cell culture supernatant was collected after 48 hours. The residual cells and debris were removed by centrifugation by 3,000×g for 15 minutes at 4℃. Transfer the supernatant to a new centrifuge tube and add 2 ml of ExoQuick-TC ™ Exosome Isolation Reagent (SBI,System Bioscience) for every 10 ml of culture medium. Then agitate the tube slightly until the sample is completely mixed. After storing at 4℃ for 12 h, centrifuge at 5,000×g for 30 minutes, discard the supernatant, and the precipitate is exosomes.
Protein was isolated by SDS-PAGE gel and transferred to PVDF membrane. After 1 h of blocking in 5% skim milk, the membrane was incubated with various primary antibodies and incubated overnight at 4℃ with Anti-Alix (1: 1000, Santa Cruz), Anti-α-SMA (1: 1000, Abcam), Anti-CD63 (1:2000, Abcam), Anti-FAP (1: 1000, Abcam), Anti-ICAM1 (1: 1000, Abcam), Anti-MMP9 (1: 1000, Abcam), Anti-PDGFR beta(1: 1000, Abcam), Anti-β‐actin(1: 1000, Sigma), Anti-E-cadherin (1: 1000, Sigma), Anti-N-cadherin(1: 1000, Sigma), Anti-Vimentin(1: 1000, Sigma), Anti-snail (1: 1000, Sigma), Anti-Zeb1(1: 1000, Sigma), Anti-Zeb2(1: 1000, Sigma), Anti-Slug(1: 1000, Sigma), Anti‐HSP70 (1: 1000,Cell Signaling Technology), Anti‐GM130(1: 1000, Sigma), Anti-ZRANB2(1: 1000, Affinity), Anti-ACO1(1: 1000, Affinity), Anti-MBNL1(1: 1000, Affinity), Anti-TCEAL7(1: 1000, Affinity). And after three times of elution with TBST the secondary antibody linked with horseradish peroxidase was incubated for 1 hour and the electrochemical luminescence was observed by chemiluminescence apparatus. Results WB quantization was performed by ImageJ software.
Electron Microscopy (EM)
The separated exosomes were fixed with 4% paraformaldehyde and then dripped into the formaldehyde-coated electron microscope grid and fixed with 1% glutaraldehyde for 10 minutes. The sample was stained with 1% uranyl-oxalate solution for 5 minutes. Removing excess liquid and images were obtained using JEOL 1011 transmission electron microscope at 60kV.
A lentiviral plasmid encoding miR-106a/mimics or inhibitor, TCEAL7 and its siRNA and negative control was designed and produced by Genechem (Shanghai, China). Lipofectamine 2000 reagent (Invitrogen, California, USA) was used to transfect breast cancer cells according to the instructions. GW4869 (Sigma, California, USA) was used to inhibit exosomal release.
RNA extraction and reverse transcriptase quantitative real-time PCR (qRT-PCR)
All operations were performed according to the manufacturer's instructions. TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc) isolated total RNA from cells and exosomes, and the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc) was used for cDNA synthesis. The expression of mature miRNAs was detected by real-time quantitative PCR (qPCR) using SYBR Premix (Perfect Real Time) kit. Transcripts of U6 act as internal parameters to standardize RNA input. Determining analysis and fold changes using the comparative threshold cycle (Ct) method.
Meta-analysis of global gene expression data in the Oncomine database (Compendia Bioscience, Ann Arbor, USA) was performed using primary filters for “breast cancer” and “cancer vs normal analysis”, data type filter to use “mRNA” data sets and sample filter to use “clinical specimens” (10 datasets representing 2941 patients). Patients include patients of all ages, genders, disease stage or treatment. Data were acquired in an unbiased manner by compiling all the Oncomine studies with significantly altered TCEAL7 expression at the threshold settings (P-value = 0.05, foldchange = 2, and gene rank = all). Significant studies, in which at least one analysed group was comprised of three patients or less were excluded. In the Oncomine database, all data are reported as log2 median-centered intensity. Export the dataset from Oncomine and analyze it in GraphPad Prism V7 software.
Migration and invasion
In vitro invasion experiments were performed by 6.5mm Transwell 24-well plates. Add 600 µL of RPMI-1640 medium containing 10% FBS in the lower chamber, and coat the upper chamber with 25 µL of Matrigel Basement Membrane Matrix (BD) and serum-free RPMI-1640 mixed solution (configure scale bit 1: 6), and dry at 37°C at least 4 h. Then implant 120,000 cells in the upper chamber and incubate for 16 h in the incubator. Wash three times with PBS, fix with 4% paraformaldehyde, and stain Giemsa for 15 minutes. Rinse off the staining solution with slow running water and leave to dry. Scanned under a microscope after sealing with resin.
DiD Labeling of Exosomes
Vybrant DiD (Life Technologies) was used to label exosomes according to the manufacturer's instructions. In brief, DiD and PBS were diluted at 1:1000 and incubated at room temperature for 10 minutes. Exosomes were then re-purified using a qEV Size Exclusion Columns (Izon Science Ltd.).
The cells were fixed with 4% paraformaldehyde, 0.5% TritonX-100 was infiltrated for 15 min, and washed three times with PBS. Blocking with BSA for 1 hour, and incubate at 4°C with the primary antibody for 1 hour in the dark, then incubate with Alexa Fluorescence 568 (1: 1000, Invitrogen) at 37°C for 1 hour in the dark with the secondary antibody, stained with DAPI (1: 5000, Sigma). Finally, the cells were observed with a confocal fluorescence microscope.
The 3′UTR region (5′-GCUUUCUGUUUGUCUGCACUUUC-3′) of TCEAL7 might be targeted by miR-106a was screened by TargetScan software. 3′UTR terminal mutation reporting vector was designed and constructed by Shanghai GenePharma Co.,Ltd. According to the instructions of Lipofectamine2000, the luciferase reporter vector was used to co-transfected MDA-231 cells with miR-106a, miR-106a inhibitor, miR-106a mutant and control sequence, and then the cells were put into an incubator at 37°C and 5%CO2 for 48 h. Luciferase activity was tested by detector.
The tissue was embedded in paraffin fixed with formalin, dewaxed with xylene and dehydrated with ethanol, and then extracted with 0.01mm citrate buffer (pH6.0). The Rabbit anti-SMA monoclonal antibody was incubated overnight at 4℃, and anti-rabbit secondary antibody was incubated at 37℃ for 30 minutes. Finally, it was developed with DAB and counterstained with hematoxylin. Taking photos under an optical microscope.
Immuno-precipitation using anti-ZRANB2 antibody was performed at 48 h after treatment. Briefly, cells were collected and lysed by the lysis buffer. Then Lysates were centrifuged at 14,000×g for 15 min. The supernatant was mixed with ZRANB2 antibody (Affinity) overnight at 4°C, and then co-cultured with beads (santa cruz) for incubated at 4°C for 4 h. The beads were washed five times in lysis buffer and then subjected to Western blot (WB) analysis.
In vivo tumour growth and metastasis experiment
The prepared 10 mice were divided into two groups. One group of MDA-231 cells were floating in 150 mL of PBS and injected into the mice through the tail vein, and the other group was injected with cancer cells co-cultured with exosomes miR-106a. Thirty days later, the tumor growth was monitored using biofluorescence imaging technology, and then the mice were sacrificed, and the lung tissue was removed for observation and photographing. All experimental procedures involving mice were performed in accordance with the Guidelines for the Care and Use of Experimental Animals and were approved by the Animal Care and Use Research Committee of Weifang Medica University.
All data were analyzed by SPSS 17.0 statistical software. Each experiment was repeated for 3 times, and the comparison of measurement data was conducted by independent sample t test and paired sample t test. Mean ± SD was used for quantitative data, and P < 0.05 was considered statistically significant.