The experiments were approved by the Institutional Review Board of the 3rd affiliated hospital of Zhengzhou University. All participants provided written informed consent. Efforts were made to avoid all unnecessary distress to the animals.

General information of patients

The tissue samples of 79 MB patients were collected from the 3rd affiliated hospital of Zhengzhou University. The median age at diagnosis for MB patients was 9.94 years. Inclusion criteria: complete clinical data; MB confirmed via the pathologic diagnosis; no radiotherapy or chemotherapy before surgery. Exclusion criteria: incomplete clinical data; other primary tumors. Another 20 cases of normal cerebellar tissues were taken as a control via surgery excision after cerebellar hemorrhage.


Paraffin-embedded sections were deparaffinized in xylene and rehydrated in 100, 95, 90, 85, and 75% gradient series of ethanol. Then, the antigen was repaired in a citrate buffer (pH 6.0) at 120 °C, the endogenous peroxidase activity was blocked with 3% H2O2, and the sections were incubated with WDR82 primary antibody (1:200, Abcam, MA, USA) overnight and with the secondary antibody (1:500; Abcam). Diaminobenzidine-developed sections were counter-stained with hematoxylin solution.

Extraction and induction of bone marrow-derived macrophages

BALB/c mice (Henan Experimental Animal Center, Henan, China) aging 6–8 weeks, weighing 18–20 g, were euthanized. The femur and tibia were taken, and the bone marrow was syringed by serum-free Roswell Park Memorial Institute (RPMI) 1640 medium and filtrated with a 70-μm mesh. The filtrate was centrifuged and lysed with 5 mL erythrocyte lysis for 5 min. The monocytes obtained were stimulated in RPMI 1640 medium containing macrophage colony-stimulating factor (M-CSF, Peprotech, 25 ng/mL) for 72 h, 48 h, and 24 h (the medium was renewed at each time point), thus to differentiate to M0 macrophages.

To generate M2 macrophages, M0 macrophages were incubated with 20 ng/mL interleukin (IL)-4 (Peprotech) and 20 ng/mL IL-13 (Peprotech) for 24 h. Cell morphology was observed under an inverted microscope, and M2 macrophages-related markers arginase1 (AGR1) and CD206 were analyzed by Western blot.

M2 macrophage transfection and exosome purification and characterization

Isolated M2 macrophages were transfected with miR-155-3p mimic or mimic NC (Ribobio, Guangzhou, China) via lipofectamine 3000 (Invitrogen, CA, USA). The ultracentrifugation method was used to extract exosomes from M2 macrophages transfected with miR-155-3p mimic or mimic NC and named Exo, Exo-miR-155-3p mimic and Exo-mimic NC accordingly.

Exosomes were isolated from the culture supernatant of M2 macrophages with ultracentrifugation method. The collected culture supernatant was centrifuged at 500×g and at 2000×g to remove the cell precipitation and cell debris. The obtained solution was filtrated via a 0.22-μm membrane and centrifuged at 1,00,000×g. The precipitation was re-suspended with PBS and centrifuged at 1,00,000×g to obtain exosome precipitation. During this process, the temperature of the sample never fell below 4 °C. The quantity of exosomes was measured using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Western blot, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to identify M2-Exo.

Fluorescence microscopy analysis of exosome internalization

CM-Dil (2 μL, Sigma-Aldrich, MO, USA) was mixed with 100 μg exosomes, and resuspended in 18 mL PBS for 2-h centrifugation (1,20,000×g). The pellet was resuspended in 20 mL PBS and centrifuged at 1,20,000×g for 2 h. Then, the pellet was resuspended in 200 μL PBS and incubated with cells for 24 h. After fixation with polyformaldehyde, cells were observed under a fluorescence microscope.

Cell culture and treatment

In MB cell lines Daoy, D283, ONS-76, D341 and human glial cells (Shanghai YaJi Biological, Shanghai, China), STR identification and mycoplasma detection were performed.

Daoy cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin mixture. Cells with 60% confluence were transfected using Lipofectamine 3000 (Invitrogen), and collected at 48 h after transfection for further experiments. Daoy cell line was transfected with miR-155-3p mimic (10 nM), mimic NC (10 nM), miR-155-3p inhibitor (25 nM), inhibitor NC (25 nM), miR-155-3p mimic (10 nM) + WDR82 overexpression plasmid (1 μg/mL), and miR-155-3p mimic (10 nM)  +  empty plasmid (1 μg/mL), respectively. All the oligonucleotides or plasmids were obtained from Ribobio (Guangzhou, China).

Five μg of Exo, Exo-miR-155-3p mimic and Exo-mimic NC were co-cultured with 1 × 105 Daoy cells for 48 h when the cell confluence was 60%. Cells were collected for further experiments.

Proliferation assay

Transfected Daoy cells or Daoy cells treated with M2-Exo were cultured in 96-well plates, and analyzed by CCK-8 (Dojindo, Japan). The optical density450 nm value was detected on the micro-plate reader at 24 h, 48 h and 72 h.

Colony formation assay

Transfected Daoy cells or Daoy cells treated with M2-Exo were seeded in 6-well plates with 200 cells/well [22]. The experiment was terminated 2 weeks later when the colonies were visible. The cells were fixed with anhydrous methanol solution, stained with 0.1% crystal violet solution, and photographed. The number of colonies was counted using the Image J software.

Cell invasion assay

A 24-well Transwell plate (Corning) pre-coated with Matrigel (BD) was used for cell invasion. Transfected Daoy cells or Daoy cells treated with M2-Exo (4 × 104) were seeded into the upper chamber. The medium in the upper chamber was FBS-free DMEM, and the medium in the lower chamber was DMEM supplemented with 10% exosomes-free FBS. After 24 h, cells transferred to the lower chamber were stained with 0.5% crystal violet and photographed under a microscope (Nikon, Japan).

Scratch test

Transfected Daoy cells or Daoy cells treated with M2-Exo were seeded with 5 × 104 cells per well into 24-well plates. After cell adherence, a straight line was drawn on the monolayer cells using a 10 μL pipette. Cells were cultured with FBS-free DMEM (500 μL/well) for 24 h and observed under an inverted microscope.

Flow cytometry

Transfected Daoy cells or Daoy cells treated with M2-Exo were seeded into 6 well plates with 1 × 106 cells/well. After culturing for 12 h, cells were resuspended with 100 μL buffer. According to the protocol of Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Beyotime), 10 μL FITC (50 mg/L) and 5 μL propidium iodide(50 mg/L) were added to the cultured cells. Then, cells were added with 200 μL binding buffer and loaded to the flow cytometer (FACSCalibur, BD Biosciences, NJ, USA).

In vivo experiment

BALB/c nude mice of specific pathogen-free grade, aging 3–4 weeks, were purchased from Henan Experimental Animal Center. To observe the effect of miR-155-3p on tumor growth in MB, Daoy cells (1 × 106) transfected with miR-155-3p overexpression lentivirus, miR-155-3p low expression lentivirus were suspended in 100 µL PBS and injected subcutaneously in the left groin of nude mice. To observe the effect of M2-Exo on the growth of MB, Daoy cells (1 × 106) were subcutaneously injected into the left groin of nude mice. Meanwhile, 5 mg exosomes (Exo, Exo-miR-155-3p mimic and Exo-mimic NC) were administered into mice via tail vein injection once every 3 days for 2 weeks. The tumor volume (V  = 1/2 × L  ×  W2, L  =  tumor length, W  =  tumor width) was measured every 7 days with a vernier-caliper. A tumor growth curve was drawn and all nude mice were euthanized after 28 days.


Total RNAs were isolated form tissues, cells, exosomes using Trizol (Invitrogen), and A260/A280 was determined by an ultraviolet spectrophotometer. RNA concentration (μg/μL)  =  (A260 × 40 ×  dilution factor)/1000. The purity should be 1.8–2.1. For WDR82, cDNA was collected from RNA (2 μg) through first-strand cDNA synthesis kit (Thermo Fisher Scientific) while for miR-155-3p, that was collected through NCode miRNA first-strand cDNA kit (Invitrogen). Real-time PCR was performed on the ABI7900 PCR system (Applied Biosystems, CA, USA) using SYBR Green PCR Master Mix (Takara, Dalian, China). The loading control of miR-155-3p was U6, and that of WDR82 was β-actin. The relative expression was calculated by 2Ct method and the primer sequences are shown in Additional file 1: Table S1.

Western blot analysis

The total protein was extracted from tissues, cells and exosomes using modified RIPA buffer and sonication, and the protein concentration was detected by BCA method. Total protein extract (20 µg) or exosomal protein (10 µg) was separated using a 10% or 15% polyacrylamide gel and transferred to a 0.22-μm polyvinylidene fluoride membrane (Merck Millipore, USA). The membrane was blocked with 5% skim milk for 1 h, and incubated with the primary antibodies WDR82 (1: 100), β-actin (1: 1000, Abcam), CD206 (1: 1000, R&D Systems, Minneapolis, MN, USA), ARG1 (1: 1000, Proteintech, Chicago, USA), CD81 (1:1000), Alix (1: 1000), TSG101 (1: 1000), GRP94(1:500) (Santa Cruz Biotechnology, CA, USA) overnight at 4 ℃, and the corresponding secondary antibody for 1 h. The membrane was reacted with enhanced chemiluminescence solution for 5 min and detected in the exposure apparatus.

Dual luciferase reporter gene assay

The luciferase reporter assay was carried out using pmiR-RB-REPORT vector (RiboBio) containing the wild-type (WT) or mutant (Mut) WDR82 3′-UTR sequences. miR-155-3p mimic or the corresponding controls along with the WT/Mut WDR82 3′-UTR vectors was transfected into Daoy cells using Lipofectamine 3000 (Invitrogen). At 48 h after transfection, the dual luciferase assay kit (Beyotime) was used to measure luciferase activity; the luciferase activity was standardized to Renilla luciferase activity.

Statistical analysis

Statistical analyses were performed with SPSS 21.0 (IBM, Chicago, IL, USA) and Graphpad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Data were presented as mean  ±  standard deviation (SD). Student’s t test was applied to evaluate the significance between two samples. analysis of variance (ANOVA) was used for comparison among multiple groups, and Tukey’s post hoc test was applied for pairwise comparison after ANOVA. Correlation analysis was conducted by Pearson test. The correlation between miR-155-3p expression and the clinicopathological characteristics of patients with MB was determined via chi-square test or Fisher’s exact test. Predictors were kept if they were significant at a P value of 0.05 or smaller.

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