Patient enrollment and sample preparation
All participants were enrolled through the Genetic investigation of Inherited and Familial Tumor Syndrome (GIFTS) study between January 2018 and June 2018 from the Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS). Patients were eligible for enrollment if they had an evident histologic diagnosis of breast cancer and without distant metastasis. Age-matched healthy women were also recruited as a control group. Peripheral blood samples of 10ml from these breast cancer patients and ten age-matched healthy women were collected at CHCAMS. Blood samples were collected in vacuum tubes with EDTA and centrifugated at 3,000 × g for 15 minutes at 4℃, the collected supernatant (5 ml plasma) was preserved at -80 °C before use. All participants signed written informed consent. Ethics approval for the study was obtained from the Research Ethics Committee of CHCAMS.
Exosome isolation
The collected plasma was thawed at 37℃ and then centrifugated at 3,000 ×g for 15 minutes to remove cell debris. Aspirated supernatant was diluted 7-fold with PBS and centrifuged at 13,000 ×g for 30 minutes [19]. Large particles were removed using 0.22 μm filters. Then the collected supernatant was ultra-centrifuged at 100,000 ×g, 4℃ for 2 h (CP100NX; Hitachi, Brea, CA, USA). The pellet containing exosomes was re-suspended in PBS and ultra-centrifuged again at 100,000 ×g 4℃ for 2 h. The isolated exosomes were re-suspended in 100 µl PBS after PBS washing for further analysis.
Exosomes Characterization
The nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot analysis using rabbit polyclonal antibody CD63, TSG101, and calnexin were conducted following the previously reported protocols [20].
Exosomal RNA isolation and RNA analyses
The RNAs were extracted from plasma-isolated exosomes using the miRNeasy® Mini kit (Qiagen, cat. No. 217004). RNA yields as well as DNA contamination were monitored on a 1.50% agarose gel. The NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, Germany) was used to assess RNA concentration and purity. The integrity and distribution of RNAs were analyzed using the Agilent Bioanalyzer 2100 system with RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA).
Library preparation and sequencing
First, a total amount of 5 ng RNA per sample was depleted of rRNA using the RiboZero magnetic kit (Epicentre, Madison, Wisconsin, USA). Then, the sequencing libraries were generated using the Ovation® RNA-Seq System (NuGEN, CA, USA). A total amount of 2.5 μg RNA per sample was used as input material for sample preparation of small RNA libraries. Then, the libraries were generated using the NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, USA). The index codes were added to attribute sequences to each sample. Finally, the polymerase chain reaction (PCR) products were purified using the Agencourt Ampure XP system (Beckman Coulter, Brea, CA, USA). The the library quality was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) and quantitative PCR. The cluster of the index-coded samples was generated by the acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumina, San Diego, CA). At last, the sequencing was performed on the Illumina Hiseq platform using the library preparations and paired-end reads were generated.
Quantitative differential expression analysis of miRNA
The sequence alignment was performed using the Bowtie tool [21] with several databases including the Silva database (https://www.arb-silva.de/), the GtRNAdb database (http://gtrnadb.ucsc.edu/), the Rfam database (http://rfam.sanger.ac.uk/), and the Repbase database (http://www.girinst.org/) [22]. Subsequently, the rRNA, transfer RNA, small nuclear RNA, small nucleolar RNA, and other non-coding RNA were filtered. Then, the miRNA including known miRNAs and novel miRNAs were detected using the remaining reads, in which the novel miRNAs were predicted according to the miRbase database and Human Genome (GRCh38), respectively. Read counts of the miRNAs were generated from the mapping results and have been standardized as the total mapped reads (TPM) per million. Circulating exosomal miRNA profiles of samples with two conditions were compared by the Student’s t test (two-tailed), and each miRNAs with a log2|fold change|>0.58 and p <0.05 was considered as differential expression. Hierarchical clustering was performed with R package ‘pheatmap’ using the ward.D2 method.
Cell culture and transfection
The MCF-7 cell line was cultured at 37℃ with 5% CO2. Dulbecco's modified Eagle's medium (DMEM, SH30022.01, HyClone, South Logan, UT, USA) with 10% fetal bovine serum (FBS, FND500, ExCell Bio., Shanghai, China) was applied as culture medium. One hundred units per milliliter penicillin and 100 μg/ml streptomycin (SV30010, Hyclone, Logan, UT, USA) were also added into DMEM. Until the density reached approximately 50-70%, cells were transfected for 48 hours with miR-363-5p mimic, mock negative control (NC), miR-363-5p inhibitor or inhibitor NC (Ribo, Guangzhou, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
RNA extraction and quantification
The miRNA was extracted from MCF-7 cells with the miRcute miRNA isolation kit (DP501, Tiangen, China). Total RNA was extracted from transfected MCF-7 cells with the total RNA rapid extraction kit (China (220010, Feijie biological, China). After quality control, the FastQuant RT kit (KR106, China) was used to reverse transcribe the miRNA or RNA sample into cDNA. qRT-PCR was performed in an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, California, USA). SuperReal PreMix Plus (SYBR Green) mixture (FP205, Tiangen, China) was applied for reactions. The relative amount of miR-363-5p to control U6 and PDGFB to control GAPDH transcripts were analyzed by the 2-ΔΔCt method. Primers applied were listed as follows: miR-363-5p: forward: 5’-CGGGTGGATCACGATG-3’; reverse: 5’-CAGTGCAGGGTCCGAGGTAT-3’; U6: forward: 5’-CTCGCTTCGGCAGCACA-3’; reverse: 5’-AACGCTTCACGAATTTGCGT-3’ [23].
Cell proliferation assay
The MCF-7 cells were planked in 96-well plates with a density of 5 × 103 cells/well. The proliferation of cells at 0h, 24h, 48h, and 72 h after transfection was examined using the CCK-8 proliferation assay kit (MA2018-L, Meilunbio, China). Ten microliters of CCK-8 reagent was added to the medium at every time phase. Absorbance at 450nm was measured after 3 h incubation using microplate spectrometer reader (Molecular Devices, CA, USA).
Transwell migration assay
After transfection with miR-363-5p or NC for 48 h, MCF-7 cell was washed twice with FBS-free medium, and then re-suspended in FBS-free medium at a density of 1×105 cells/ml. Transwell chamber (pore size 8.0 μm, 3422, Corning Costar, MA, USA) pre-treated with the FBS-free medium was placed in a 24-well plate. After removing the pre-treatment medium, the lower chamber was added with 600 μL 10% FBS containing medium, while the upper chamber was added with 100 μL cell suspension. After incubating for 48 h, the chambers were fixed and stained with methanol and 0.2% crystal violet. After staining, cells on the chamber surface were removed carefully with water and cotton swabs. The number of perforated cells in the outer layer of the basement membrane of each chamber (migrating cells) was counted in five random high power fields with phase-contrast microscope (NIB-100F, Novel, China).
Statistical analysis
Analyses were performed with R Statistical Software (version 3.5.3). Pre-set p<0.05 was defined as statistically significant. Quantitative data were measured as mean ± standard deviation. The comparison of mean values between two groups was analyzed using Student’s t test and Mann-Whitney U-test. Pearson’s test was used to evaluate the exosome-tissue miRNA correlation and miRNA-target mRNA correlation. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance, and the area under the curve (AUC) were calculated with R package ‘ROCit’ [24]. Kaplan Meier method and log-rank test were applied to compare survival differences using R package ‘Survival’.