Introduction
Medulloblastoma (MB) is a most prevalent malignancy in children [
1]. The occurrence of MB is the highest in brain tumors, while its prognosis is much unpleasing [
2,
3]. MB is assigned into four main molecular subgroups, including wingless, sonic hedgehog, Group 3 and Group 4, each with mutational and transcriptomic signatures, clinical outcomes and apparent cytogenetics [
4]. Risk factors of MB consist of radiation, age at diagnosis, female sex and additional unknown elements [
5]. High-dose chemotherapy, surgery resection and radiation to the primary tumor site and craniospinal axis are the universal therapeutic approaches for MB [
6]. However, the 5-year overall survival rate of patients with MB is around 65% [
7], and the survivors have to endure neurological, cognitive and endocrine disorders resulted from the aggressive therapy [
8]. Therefore, it is essential to develop more feasible and effective therapeutic strategies for MB.
MicroRNAs (miRNAs) control biological processes [
9]. A previous study has demonstrated that miR-155 takes on a crucial role in accelerating glioma progression and offers prognostic values [
10]. In addition, miR-155-3p suppression could reduce glioma cell growth and proliferation in a mouse model and elevates the survival of mice with gliomas [
11]. Macrophages are inborn immune cells that could modify different kinds of immune responses, antigen presentation and phagocytosis. After differentiation from their monocyte precursor, macrophages polarize toward M1-like or M2-like phenotype [
12]. Exosomes, an important form of extracellular vesicles, have been confirmed as the intercellular communication mediators in various physical processes related to cell proliferation and migration [
13]. Exosomes can be defined by several common characteristics, including size, density, morphology, and certain enriched protein markers [
14]. Exosomes can transfer macrophage phenotype to M2 activated macrophages [
15]. It has been suggested that exosomes are involved in MB tumor biology, such as stimulation of tumor cell proliferation and migration [
16]. In the present study, we first characterized the exosomes to determine their size, appearance and expression of protein exosomal markers, in accordance with recommendations by minimal information for studies of extracellular vesicles 2018 (MISEV2018) [
17], and then explored that whether M2 macrophages-derived exosomes (M2-Exo) could load miR-155-3p to mediate the progression of MB cells. M2-Exo display high miR-155 expression, and M2-Exo-mediated tumor growth is partly depended on miR-155 [
18,
19]. WD repeat domain 82 (WDR82) is a c-terminal domain-combination protein recruiting the Setd1A Histone H3-Lys4 methyltransferase complex [
20]. It has been accepted that WDR82 reduction is correlated with a poor prognosis of patients with colorectal cancer (CRC) [
21], while the relationship of WDR82 with MB was little studied.
Therefore, the aim of this study was to investigate the role of miR-155-3p-loaded M2-Exo in MB progression by regulating WDR82.
Materials and methods
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.
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.
Immunohistochemistry
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.
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.
RT-qPCR
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 2
−△△Ct 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.
Discussion
MB is a high-ranking malignancy recognized as a member of childhood cerebellum tumors [
23]. Meanwhile, the pathophysiological basis of MB still remains poorly understood. Exosomes, virus-sized membrane vesicles produced extracellularly from cells may exhibit roles in MB pathogenesis but are as yet largely studied in this disease. For these reasons, this study was to investigate the role of the transfer of miR-155-3p by M2-Exo in promoting MB progression by targeting WDR82.
The observation of the study was that M2-Exo promoted the proliferation, colony formation, invasion and migration abilities, inhibited apoptosis in vitro, and elevated the tumor growth rate in nude mice in MB. Indeed, it has been clarified that exosomes are involved in the pathogenesis of MB [
16]. Significantly, exosomes from human gastric epithelial cells or serum exosomes from infected patients and mice clearly reduce endothelial functions with decrease of migration and proliferation [
24]. This is consonant with the fact that human bone marrow mesenchymal stem cell-derived exosomes facilitate osteosarcoma cell invasion, proliferation and migration [
25]. Furthermore, it has been observed that M2-Exo could induce colon cancer cell proliferation and invasion and reduce apoptosis [
26]. Also, Yuan et al. have described that M2-Exo perform aggressively during the progression of oral squamous cell carcinoma cell growth through acting as a carrier of miR-31-5p [
27].
Our study revealed that miR-155-3p was up-regulated in MB tissues of patients and was correlated with the molecular subtype classification of MB. It has been found that miR-155 suppression may be a functional anti-tumor choice for glioma [
10]. In addition, miR-155 is overexpressed in nonfunctional pituitary adenomas samples and adenomas [
28] and in HCC [
29]. Meanwhile, we presented that inhibition of miR-155-3p reduced proliferation, colony formation, invasion and migration abilities and promoted apoptosis of MB cells in vitro. According to a report by Tao et al. it is suggested that silencing of miR-155-3p restrains the proliferation, invasion and migration of clear cell renal cell carcinoma cells [
30]. Moreover, it has been specified that miR-155-3p up-regulation in breast cancer cells blocks cellular apoptosis [
31]. On the other hand, miR-155-3p up-regulation in CRC could reduce the levels of WDR82, involving in the promotion of tumor progression [
32]. Besides, miR-155-3p could induce the polarization of M2 macrophages and promote the progression of glioma [
33].
Some new findings were observed in this study that WDR82 was down-regulated in MB tissues of patients and cell lines; and WDR82 overexpression reversed the promoting effects of miR-155-3p up-regulation on MB cell progression in vitro and in vitro. In the area of CRC, it is described that WDR82 is lowly expressed in cancer patients, and knockdown of WDR82 is related to shortend overall survival and poorer outcomes [
21]. As to the role of WDR82 in the aggressiveness of tumors, Lei et al. have discussed that WDR82 downregulation enhances the growth of lung cancer cells [
34].
In conclusion, the study stresses that inhibited miR-155-3p-loaded M2-Exo repress MB cell progression through down-regulating WDR82, which might offer a deep insight on MB-related mechanism and molecule-based mechanism may be a possible target for future therapies of MB. More researches should be under taken to verify the relationship of miR-155-3p with WDR82 in MB.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.