Database analysis
miRCancer is a publicly available database (http://mircancer.ecu.edu/) that provides a complete set of miRNA expression profiles in various human cancers. This information is automatically extracted from Pubmed. Additionally, DbDEMC (database of Differentially Expressed MiRNAs in human Cancers) is an integrated database containing high-throughput data for differentially expressed miRNAs in human cancers. DbDEMC 2.0 is also publicly available at http://www.picb.ac.cn/dbDEMC. The CGGA database (publicly available at http://cgga.org.cn/index.jsp) is a user-friendly web application for data storage and analysis of brain tumour datasets from over 2000 samples of Chinese cohorts.
Cell culture and selection
The human normal glial cell line HEB, as well as the human glioma cell lines U87, U251, A172, LN18, SHG-44, and U138 were purchased from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat DNA profiling. Primary glioma cells, G15, were isolated from tumour tissues of glioma patients, as previously described [26]. G15 cells were derived from grade IV astrocytoma. All cells were cultured in high-glucose Dulbecco’s modified eagle medium (DMEM; Gibco, Grand Island, USA) supplemented with 10 % foetal bovine serum (FBS; Gibco, Grand Island, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin. The cells were maintained in a humidified chamber containing 5 % CO2 at 37 °C. The medium was replaced three to four times per week. Cell passaging was carried out when cell confluence reached approximately 80 %. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-375 in the six glioma cell lines, and the two cell lines with the lowest expression of miR-375 were selected for subsequent experiments. Meanwhile, the primary glioma cell line, G15, was also selected for subsequent experiments.
Preparation of lentiviral vector and transfection
MiR-375 mimic and miR-control were purchased from GenePharma (Shanghai, China). MiRNA transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA), according to the manufacturer's instructions. The extent of overexpression was evaluated by qRT-PCR 24 h after transfection. MiR-375 mimic and miR-control lentiviruses were synthesised by Genechem (Shanghai, China). For stable overexpression of miR-375 in glioma cells, miR-375 mimic or miR-control lentiviruses were added to cells, according to the manufacturer's protocol. After 24 h, clones with stable expression were selected by incubating cells for 3 weeks with 5 μg/mL puromycin (Sigma, St Louis, USA) in complete medium containing 10 % FBS. The sequences of miR-control and miR-375 mimic were 5ʹ-TTCTCCGAACGTGTCACGT-3ʹ and 5ʹ-TTTGTTCGTTCGGCTCGCGTGA-3ʹ, respectively.
Treatment of cells with CTGF or epidermal growth factor (EGF)
After miR-375 was overexpressed in glioma cells, the cells were treated with 200 ng/mL CTGF (Peprotech, Rocky Hill, USA) or 20 ng/mL EGF (Abcam, Cambridge, UK) for 24 h.
Cell counting kit assay
The glioma cells were seeded in 96-well plates (Costar, Cambridge, USA) at a density of 3 × 103 cells/well, and cultured at 37 °C for 3–5 days. Viable cells were analysed with the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s guidelines using a microplate reader (BioTek, Winooski, USA) at 450 nm.
5-ethynyl-2ʹ-deoxyuridine (EdU) cell proliferation assay
The rate of cell proliferation was measured using an EdU cell proliferation assay kit (KeyGEN BioTECH, Nanjing, China), according to the manufacturer’s protocol. The glioma cells were incubated with 250 μL of EdU solution for 2 h at 37 °C, and then fixed in 4 % paraformaldehyde for 15 min, permeabilised with 0.4 % Triton X-100 (Sigma, St Louis, USA) for 10 min, and incubated with Apollo®reagent (250 μL) for 30 min. Subsequently, the nuclei were stained with 4ʹ,6-diami-dino-2-phenylindole (DAPI; Sigma, St Louis, USA) for 30 min, and images were obtained using an inverted fluorescence microscope. The proportions of Edu-positive and DAPI-positive cells were then calculated.
Wound healing assay
At least five transverse lines were drawn on the back of each well of a 6-well plate using a marker pen. Next, 5 × 105 cells were added to each well and incubated overnight. Vertical lines were then drawn using a pipette tip. After removal of the detached cells, serum-free medium was added, and the cells were incubated in culture with 5 % CO2 at 37 °C. Finally, the cells were photographed at 0, 24, and 48 h.
Transwell migration and invasion assays
The migration and invasion assays were performed using cell culture inserts with 8 µm pores and 24-well plates (Costar, Cambridge, USA). For the invasion assay, the upper chamber was coated with 50 μL of Matrigel (BD Biosciences, San Jose, USA). To assess migration, the filters were not precoated with Matrigel. The glioma cells were added to the top chamber in serum-free medium. The bottom chamber was filled with 10 % FBS DMEM. After 24 or 48 h of incubation, the cells in the top chamber were removed using a cotton swab, and the membrane was fixed in 4 % paraformaldehyde for 15 min, and stained with Crystal Violet for 15 min. Images of five random fields were taken for each well, and quantification was performed by using ImageJ (NIH, Bethesda, USA).
Bioinformatic analysis of miRNA
The TargetScan (http://www.targetscan.org), Pictar (https://pictar.mdc-berlin.de/), miRanda (http://www.microrna.org/microrna/home.do), and StarBase (http://starbase.sysu.edu.cn/index.php) algorithms were used to identify putative targets of miR-375.
RNA extraction and qRT-PCR
Total RNA from glioma cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, USA). Exosome RNA extraction was conducted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). The PrimeScriptTMRT reagent kit and the gDNA Eraserkit (TaKaRa, Tokyo, Japan) were used to reverse transcribe 1 μg of total RNA into complementary DNA. An SYBR® Premix Ex TaqTM kit (TaKaRa, Tokyo, Japan) was used for qRT-PCR on a LightCycler 480 instrument (Roche, Indianapolis, USA). The relative RNA expression was determined by the comparative Ct (2-ΔΔCt) method. The primers were provided by Sangon Biotech Ltd. Company (Shanghai, China; Table 1).
Western blot analysis
Total and exosomal proteins were extracted using the Whole Cell Lysis Assay (KeyGEN BioTECH, Nanjing, China). Protein extracts were separated by 8–12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, USA). After blocking with 5 % BSA (Sigma, St Louis, USA), the membranes were incubated with primary antibodies (Supplementary Table S1) overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The protein bands were visualised by enhanced chemiluminescence (ECL; Millipore, Bedford, USA), while protein band intensities were analysed by ImageJ software and normalised to GAPDH.
Enzyme linked immunosorbent assay (ELISA)
The cell culture medium was collected 72 h after treatments. CTGF secretion was detected by ELISA (Proteintech, Chicago, USA) according to the manufacturer’s instructions.
Exosome isolation
The exosomes were removed from FBS by ultracentrifugation at 100,000 × g for 8 h (Exo-free-FBS). When the confluence of glioma cell lines reached approximately 80 %, DMEM with 10 % Exo-free-FBS was added to the cells and incubated at 37 °C with 5 % CO2 for 48 h. The cell medium was then collected and centrifuged at 4 °C and 300 × g for 10 min. The supernatant was collected and recentrifuged for 15 min at 2000 × g and 4 °C and for another 15 min at 5000 × g. The supernatant was then collected and centrifuged at 12,000 × g for 30 min. The final supernatant was collected and subjected to ultracentrifugation at 100,000 × g and 4 °C for 70 min (Beckman Coulter, Brea, USA). The exosomal pellets were washed with sterilised PBS and ultracentrifuged at 100,000 × g for 1 h. Subsequently, exosomes were resuspended in 100 μL of PBS and stored at −80 °C until use.
Exosome characterisation
The morphology of the isolated exosomes was examined by transmission electron microscopy (TEM; Hitachi HT7650, Tokyo, Japan). Briefly, the isolated exosomes were fixed with 2 % paraformaldehyde and spotted onto a glow-discharged copper grid on filter paper. Afterwards, the copper grid was dried for 15 min at room temperature. The samples were subsequently stained with 2 % uranyl acetate and dried for 10 min. Subsequently, the samples were examined at 100 keV. The size distribution of the isolated exosomes was analysed by a Zetasizer Nano-Zs instrument (Malvern Panalytical, Worcestershire, UK) according to the manufacturer’s instructions. The exosome concentration was evaluated using the bicinchoninic acid (BCA) protein assay kit (KeyGEN BioTECH, Nanjing, China), as per manufacturer’s instructions. For analysis of exosomal protein markers, western blotting assays with anti-CD9, anti-ALIX, anti-TSG101, and anti-calnexin antibodies (Supplementary Table S1) were performed.
Exosome secretion and uptake assay
GW4869 (10 μM; Sigma, St Louis, USA) and monensin (1 μM; MedChemExpress, Shanghai, China) were used to inhibit and stimulate exosome release, respectively. To monitor exosomal trafficking, exosomes were labelled with a PKH26 fluorescent cell linker kit (Sigma, St Louis, USA). PKH26-labelled exosomes were washed in PBS and centrifuged at 100,000 × g for 20 min at 4 °C, collected, and resuspended in PBS. PKH26-labelled exosomes were then incubated with glioma cells for 24 h. Finally, the uptake of exosomes by glioma cells was assessed by confocal fluorescence microscopy and flow cytometry.
Serum samples from patients with glioma
All blood samples were collected at the Department of Neurosurgery, Zhujiang Hospital, Southern Medical University of China. The enrolled subjects included 25 males and 16 females, with an average age of 44 years (range, 5–73). Nine of the 31 gliomas were classified as low-grade (WHO I-II), and 22 were classified as high-grade (WHO III-IV). The clinicopathological features of patients are shown in Table 2. Moreover, blood samples from ten healthy humans were extracted as controls. All blood samples were centrifuged at 3000 × g for 10 min at 4 °C for plasma extraction. Informed consent was obtained from all patients. Both the study protocol and the informed consent were approved by the Ethical Committee of Zhujiang Hospital.
Enrichment of plasma an astrocyte-derived exosomes (ADEs) for RNA extraction
For total exosome purification from plasma, 2 mL of plasma were obtained from 5 mL of blood and diluted with 10 mL of PBS. Next, the same procedure used for exosome purification from cultured cells was followed. To enrich ADEs, total exosomes were resuspended in 0.35 mL of PBS and incubated for 60 min at room temperature with 1.5 μL of mouse anti-human glutamine aspartate transporter (GLAST) biotinylated antibody (Miltenyi Biotec, Auburn, USA) in 50 μL of 3 % BSA per tube with mixing, followed by addition of 10 μL of streptavidin-agarose UltraLink resin (Thermo Fisher Scientific, Waltham, USA) in 40 μL of 3 % BSA, and incubated for 30 min at room temperature with mixing. After centrifugation at 800 × g for 10 min at 4 °C and removal of the supernatant, each pellet was resuspended in 100 μL of cold 0.05 M glycine-HCl (pH 3.0) by gentle mixing for 10 sec and centrifuged at 4000 × g for 10 min, all at 4 °C. Supernatants were then transferred to clean tubes containing 25 μL of 10 % BSA and 10 μL of 1 M Tris-HCl (pH 8.0) and mixed before addition of 700 μL QIAzol Lysis Regent. Subsequently, ADEs RNA extraction was conducted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany).
Intracranial xenograft assay in nude mice
First, to explore the role of miR-375 in vivo, U87 luciferase cells (U87-Luc; 1 × 105) were transfected with miR-375-mimic (U87-Luc-mimic) or miR-control (U87-Luc-control) lentiviruses, and stereotactically implanted into the brains of 4-week-old nude mice. Each group included eight mice (Central Animal Facility of Southern Medical University, Guangzhou, China). Second, to examine the properties of exosomes in vivo, U87-Luc-mimic cells were stereotactically implanted into the brains of 32 nude mice. On day 2 post-tumour implantation, 16 mice were randomly selected and subjected to three weekly intraperitoneal injections of GW4869 (1.25 μg/g), while the other 16 mice received three weekly injections of DMSO (the GW4869 solvent). Bioluminescent imaging (IVIS Lumina II, Caliper, USA) was used to monitor intracranial tumour growth, and the survival of the animals was recorded every day. After 3 weeks, eight mice per group were sacrificed. The brains were collected and stained with hematoxylin and eosin (H&E) and immunohistochemical (IHC). The remaining nude mice were used to generate survival curves. Meanwhile, we repeated the animal experiment with the primary glioma cell G15. The protocols used in this study were approved by the Animal Care and Use Committee of Southern Medical University.
Immunohistochemistry (IHC)
Paraffin-embedded tissue sections (4 μm in thickness) were subjected to IHC assays. Briefly, after deparaffinisation, rehydration, antigen retrieval, and blocking of endogenous peroxidase activity, the sections were exposed to specific antibodies against CTGF, Ki-67, and MMP9, (Supplementary Table S1) and subsequently incubated with appropriate secondary antibodies. Next, the signals were developed using 3,3′-diaminobenzidine (DAB; Sigma, St Louis, USA) solution, followed by counterstaining with haematoxylin (Sigma, St Louis, USA). The staining intensity was measured by ImageJ software in five random regions, and the average values were used to obtain the protein expression levels.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc, La Jolla, USA). The data were expressed as mean ± standard error of three independent experiments. The statistical significance was assessed by Student’s t-test or one-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons. The survival rates were obtained using the Kaplan–Meier method, and the differences in mortality were evaluated by log-rank-test. All tests were two-sided, and P-values < 0.05 were set as the threshold for statistical significance.