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Exosomal miRNA-21 from Toxoplasma gondii-infected microglial cells induces the growth of U87 glioma cells by inhibiting tumor suppressor genes

Toxoplasma gondii is an intracellular protozoan parasite that can modulate the microenvironment of infected hosts and is known to be associated with the incidence of brain tumor growth. In this study, we suggested that the exosomal microRNA-21 derived from Toxoplasma infection would contribute to the growth of brain tumors. Exosomes of BV2 microglial cells infected with Toxoplasma were characterized and confirmed internalization to U87 glioma cells. Exosomal miRNA expression profiles were analyzed using microRNA array and miR-21A-5p associated with Toxoplasma and tumor sorted. We also examined the mRNA level of tumor-associated genes in U87 glioma cells by changing the level of miR-21 within exosomes and the effects of exosomes on the proliferation of human U87 glioma cells. Expression of miRNA-21 was increased and anti-tumorigenic genes (FoxO1, PTEN, and PDCD4) were decreased in exosomes within T. gondii-infected U87 glioma cells. Toxoplasma-infected BV2-derived exosomes induced proliferation of U87 glioma cells. The exosomes induced the growth of U87 cells in a mouse tumor model. We suggest that the increased exosomal miR-21 from Toxoplasma-infected BV2 microglial cells may play an important role as a cell growth promotor of U87 glioma cells through a down-regulation of anti-tumorigenic genes.

More than 18.1 million common cancer cases were estimated to occur worldwide in 2018, and about 297,000 central nervous system tumors (1.6% of all tumors) are diagnosed every year 1 . Previous studies have found that risk factors for human brain tumors include various chemical products, family history, and ionizing radiation from therapeutic and diagnostic devices in the head 2 . However, the exact causes of these malignancies are yet unclear. Approximately 20% of various malignancies worldwide are due to infectious agents, including viruses, bacteria, and parasites 3,4 . Infectious agents can interfere with the host cell's genetic types of machinery, such as DNA repair and cell cycle, and can lead to chronic inflammations and immune system impairments 5 .
The infectious agents associated with human cancers are most commonly viral pathogens, including the human papillomavirus and hepatitis B and C viruses 3 . Parasites may also play potential roles in the development of human cancers 6 . Several parasite species, namely, Schistosoma haematobium, Opisthorchis viverrini, O. felineus, Clonorchis sinensis, and Hymenolepis nana are related to the development of various types of human cancers [6][7][8] .
Toxoplasma gondii is an intracellular protozoan that can modulate the microenvironment of the infected host cells 9 . This parasite was estimated to infect about 30% of people worldwide, and the entire global population is at risk 9,10 . Toxoplasma can invade vital organs, including the central nervous system (CNS), and may cause severe diseases, such as fatal meningitis and encephalitis, particularly in immunocompromised patients 9 . However, Toxoplasma can also change the environment of infected hosts to maintain asymptomatic chronic infection through modulation of the cell growth and immune responses in immunocompetent individuals 9,11 . Interestingly, several reports suggested that altered environments in the host body due to chronic Toxoplasma infection are analogous to the tumor microenvironment, considering the correlation between Toxoplasma prevalence and brain tumor incidence [12][13][14][15] . www.nature.com/scientificreports/ The exosomes are known as cell-to-cell communicators that deliver biological contents, including proteins and nucleic acids, from neighboring cells 16,17 . The exosomes can influence the tumor-related biological processes, such as anti-apoptosis, angiogenesis, and metastasis within the tumor microenvironment 18 . Especially, microRNAs (miRNAs) are small non-coding RNAs with ~ 22 nucleotides and are important post-transcriptional gene regulators controlling more than 30% of human mRNAs by miRNA-induced silencing complex (miRISC) 19 . Toxoplasma can subvert the biological processes by controlling miRNA expression in the infected hosts 20 . Host miRNAs have important clues to modulating host biological processes for the survival strategy of parasites. Therefore, the study of host miRNA profile changes in Toxoplasma infection can help us to understand the interaction between the host and Toxoplasma more clearly. Actually, Thirugnanam et al. 15 hypothesized that Toxoplasma promotes brain carcinogenesis by changing its expression on specific host miRNAs related to tumor growth and revealed that Toxoplasma could cause glioma in experimental animals.
The present study focused on the alteration of exosomal miR-21 in Toxoplasma-infected host BV2 microglial cells. We observed the possible role of altered exosomal miR-21 in the growth of U87 glioma cells through the nuclear retention of FoxO1/p27 targeted by overexpressed miR-21.

Results
Characterization of microglial cell-derived exosomes. BV2-derived exosomes were achieved using differential centrifugation and verified by various methods to prevent contamination with cellular components or other vesicles. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed different patterns between extracted proteins from BV2 cells and exosomes (Fig. 1A), and the samples were evaluated for the presence of Alix, an exosomal protein marker in western blot analysis. Alix marker was detected in the exosomal proteins but not in the BV2 cell lysate proteins (Fig. 1B). In addition, purified RNA from BV2-derived exosomes was analyzed by a bioanalyzer. The 18S and 28S nuclear ribosomal subunits were rarely observed in the RNA migra-  www.nature.com/scientificreports/ tion patterns of exosomal RNA, indicating that the purity was reliable (Fig. 1C). Finally, transmission electron microscopy revealed that the observed exosomes were shown in approximately 60-150 nm sizes with a cupshaped structure which is a typical exosome morphology (Fig. 1D).

Toxoplasma-infected BV2-derived exosomes induced proliferation and anti-apoptosis of glioma cells in vitro.
The cellular internalization of BV2-derived exosomes into U87 human glioma cells was visualized by confocal microscopy. PKH26-labeled exosomes were localized in the cytoplasm of U87 cells. The nuclei were stained with DAPI ( Fig. 2A), implying that the BV2-derived exosomes could be internalized by host cells and would influence the environment of recipient cells. After confirming the cellular internalization of BV2-derived exosomes into U87 glioma cells, we performed a cell proliferation assay to investigate the role of Toxoplasma-infected BV2 cells-derived exosomes in human glioma cell development. U87 cells were treated with exosomes of Toxoplasma-infected BV2 cells, and the results indicated that Toxoplasma-infected BV2-derived exosomes induced a significantly higher proliferation of U87 cells compared to controls (Fig. 2B).
Also, the growth of U118 cells has the same consequence as U87 in that Toxoplasma-stimulated exosomes caused the highest level of proliferation (data not shown). Based on these data, we could represent that Toxoplasma-infected BV2-derived exosomes play an essential role in the proliferation of glioma cells.

Toxoplasma-infected BV2-derived exosomes induce the growth of tumors in vivo. For investi-
gating the effect of Toxoplasma-infected BV2-derived exosomes in tumor development, we injected U87 glioma cells into nude mice for a xenograft model and administrated BV2-derived exosomes or RH-infected BV2derived exosomes to the mice. Once the tumor was apparent after 1 week, each experimental group, consisting of five mice, was divided based on tumor size for defining the same starting point, and tumor size was measured for 22 days.
In the U87 xenograft model mice, significantly higher tumor size and weight were observed in the RHinfected BV2-derived exosome group on day 22 (Fig. 3A,B). On the other hand, both the BV2-derived exosome group and the control group had no significant differences in the tumor size by treatment with exosomes. Also, the mice injected with glioma cells and exosomes exhibited the largest tumor volume visually in the RH-infected BV2-derived exosome group (Fig. 3C). These results demonstrated that Toxoplasma-infected BV2-derived exosomes induced glioma growth in a mouse tumor model.

Expression changes of exosomal miR-21a-5p (miR-21) in microglial cells infected with Toxoplasma (RH).
The data showed 37 miRNAs (16 up-and 21 down-regulated miRNAs) related to the immunity or tumor development were significantly changed in microglial cells after infection with Toxoplasma RH strain (Fig. 4A). The relative expression levels of miR-21 among altered miRNAs were validated in the BV2derived exosomes, exosomes treated with BV2 and U87 cells by real-time RT-PCR. The expression of miR-21 exhibited a significant increase in exosomes of Toxoplasma (RH strain)-infected BV2 cells (Fig. 4B). The relative expression level of miR-21 in BV2 and U87 cells was increased after the uptake of altered exosomes (Fig. 4B). The relative expression level of miR-21 was higher in the brain tissue from tumor patients and Toxoplasma (ME49

Toxoplasma-infected BV2-derived exosomes down-regulate anti-tumorigenic target gene expressions in U87 cells. Exosomes derived from RH-infected BV2 cells led to the growth of glioma
in vivo and in vitro (Figs. 2, 3). In order to discover the related mRNA, we examined the mRNA levels of antitumorigenic target genes, Fork-head box O1 (FoxO1), PTEN, and programmed cell death 4 (PDCD4) in U87 cells treated with BV2-or RH-infected BV2-derived exosomes. The bioinformatics analysis suggested that multiple tumor-related genes, including FoxO1, PTEN, and PDCD4 genes, have miR-21 binding sites 21,22 . The mRNA levels of anti-tumorigenic target genes were decreased in RH-infected BV2-derived exosomes compared to BV2derived exosomes (Fig. 5A). The FoxO1 showed a reduced protein level in RH-infected BV2-derived exosomes compared to BV2-derived exosomes (Fig. 5B). From these results, we could confirm that the down-regulated anti-tumorigenic genes by RH-infected BV2-derived exosomes support its role in tumor growth.
Changed expression of FoxO1 targeted by RH-infected BV2-derived exosomal miR-21 in U87 glioma cells. In order to know the effect of miR-21 in exosomes on tumor-related gene regulation, U87 cells were transfected with a miR-21 inhibitor using Lipofectamine 2000, and the cells were harvested 24 h after transfection. The expression levels of FoxO1 and p27 in cells transfected with the miR-21 inhibitor were compared with those treated with BV2-derived exosomes using qRT-PCR (Fig. 6A,B). The miR-21 inhibitor transfection in U87 cells significantly suppressed the FoxO1 and p27 expression (Fig. 6).

Discussion
To evade from host immune responses, Toxoplasma parasites transform themselves into tissue cysts. They are parasitic on various tissues, including the brain, heart, and skeletal muscle for the lifetime of the host, modulating the host immune responses 23 . Furthermore, they can also modulate the cell cycle and apoptosis of the host cells for their proliferation 14,24 . Toxoplasma preferentially infects host dendritic cells, neutrophils, and monocyte/macrophage lineage cells, including microglia in the brain 25 . Toxoplasma induces the differentiation of M2 phenotype macrophages, which influence wound repair after pathogen infections and is also associated with a high degree of vascularization and granuloma fibrosis 26 . Such behavioral pathogenesis of Toxoplasma infection may be linked to the related hallmarks of tumor development. The unfavorable environment modulated by Toxoplasma may be similar to appropriate precancerous conditions. Thus, we can presume that Toxoplasma infection should help develop brain tumors. Actually, it was reported that various brain tumor patients showed a high prevalence of Toxoplasma infection in sera 13 . Additionally, Toxoplasma can be another oncogenic effector and synergistically act as a helper of other infectious carcinogens developing the brain tumor. In this respect, it is noteworthy that Plasmodium falciparum and Epstein-Barr virus contributed synergistically to the formation of Burkitt's lymphoma 27 .
There are comprehensive studies on the roles of exosomes as a regulator in the cancer research area. However, the roles of exosomes between parasites and infected hosts are still poorly understood. So far, various modulating factors, including secretory proteins have explained the biological process of protozoan parasites to survive against host attacks and establish infection 16 . Recently, there has been an increasing concept that protozoaassociated microvesicles and their miRNAs communicate with host cells for promoting a favorable environment for their survival 28 . Thus, further studies are needed to find the relationship between altered exosomal miR-NAs and the proliferation of glioma cells. The changing miRNAs (miR-30c-1, miR-125b-2, miR-23b-27b-24-1, and miR-17-92 cluster genes) are regulated through promoter binding of the STAT3 in Toxoplasma-infected human macrophages and induced anti-apoptosis in response to Toxoplasma infection 29 . Toxoplasma infection can increase the expressions of miR-17-5p and miR-106b-5p, which have been implicated in several hyperproliferative diseases 30 . This evidence suggests that host miRNAs modulated by Toxoplasma infection are essential molecules in host biological behaviors for parasite survival and pathogenesis.
The altered miRNAs can affect various behaviors of the initiation and progression of malignant cells, including glioma; self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion from apoptosis, limitless replicative potential, angiogenesis, invasion and metastasis, and inflammation 31 . In glioma, altered miRNAs have been identified by several expressed profiling studies 32 .
In the present study, we confirmed the high expression level of miRNA-21 in Toxoplasma-infected host cells. The miR-21 has been identified as one of the most commonly overexpressed miRNAs in solid tumors, including glioma 33 , and its expression correlates with glioma grade 34 . Accumulating evidence suggests that miR-21 is a novel oncogene as an anti-apoptotic factor in the growth of glioma 35 and is strongly overexpressed in malignant human brain tumor tissue and plasma 36 . Interestingly, the inactivation of miR-21 in glioma cells and tissues triggers the inhibition of cell proliferation by caspase-dependent apoptosis 37 . The bioinformatics analysis of miR-21 predicted targets suggested that multiple tumor suppressor genes, including programmed cell death 4 (PDCD4), tropomyosin (TPM1), PTEN, and Fork-head box O1 (FoxO1) related to apoptosis pathways, have miR-21 binding sites 21,22,38 .  39 and can regulate tumor suppressor gene expression, such as p21, p27, Bim, and FasL 40 . The FoxO1 could bind and activate cell-cycle inhibitors, such as p27, to inhibit cell growth 41 . Moreover, FoxO1 is a crucial effector of PI3K/Akt signaling and regulates many biological processes, such as cell cycle progression and cell differentiation through the transcriptional activation of p27 42 .
Conclusively, we suggest that Toxoplasma-infected microglia cell-derived exosomal miR-21 could play an essential role as a regulator for the growth of glioma cells (Fig. 7). However, further studies are needed to find a direct relation between changed exosomal miR-21 by Toxoplasma infection and glioma growth. These findings are expected to provide a starting point of research about the relationship between Toxoplasma infection and glioma incidence.  www.nature.com/scientificreports/ To prepare tachyzoites of the ME49 strain, 20 tissue cysts were intraperitoneally injected into BALB/c mice, and at day 6 to 8 post-infection (PI), the tachyzoites converted from the bradyzoites in the cysts were harvested by washing the peritoneal cavity of infected mice with PBS. The tachyzoites of ME49 were cultured in Vero cells (monkey kidney cells, KCLB cell line; no. 10081) grown in complete RPMI (Roswell Park Memorial Institute) 1640 media (WelGENE Inc., Daegu, Korea) supplemented with 100 μg/mL of penicillin (Gibco/BRL, Grand Island, NY, USA), 100 μg/mL of streptomycin (Gibco/BRL), and 5% fetal calf serum (Lonza, Walkersville, MD, USA) at 37 °C under an atmosphere of 5% CO 2 . After culturing in Vero cells, the tachyzoites of ME49 were passed through a 25-gauge needle twice, and then debris and cells were removed by passing through 5-μm filter membranes. After washing, the tachyzoites were re-suspended in PBS 44 . The tissue cyst of T. gondii ME49 strain was maintained by injecting cysts isolated from the brains of infected C57BL/6 mice (Orient Bio Animal Center, Seongnam, Korea) intraperitoneally. The brains of mice infected with ME49 were harvested at 3 months PI and minced to isolate cysts under a microscope. Infected mice were raised under specific pathogen-free (SPF) conditions at Seoul National University College of Medicine.

Methods
RNA purification. Total RNA was extracted from BV2-derived exosomes, BV2 cells, and tissue using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, including incubation time at the elution step. The RNA concentration was determined by NanoDrop 2000 Spectrophotometer. RNA quality for microarray was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands).
Exosomes isolation. DMEM with 10% exosome-depleted FBS was prepared by ultracentrifugation at 100,000g for 16 h at 4 °C with filtration through a 0.22-µm filter (Nalgene, Rochester, NY, USA). BV2 cells, 5 × 10 5 , were cultured in DMEM with 10% exosome-depleted FBS and 1% antibiotics at 37 °C and 5% CO 2 . After 24 h incubation, the tachyzoites of RH or ME49 strain (MOI = 10) were added to the cells, and non-invaded parasites were removed in an hour and refilled with the DMEM. Exosomes from BV2 cells were isolated by modified differential centrifugation, the most widely used method. The pellet consisting of exosomes was resuspended in 300 μL of PBS for RNA or protein analysis. The concentration of isolated exosome was determined by BCA protein assay kit (Pierce, Rockford, IL, USA) and NanoDrop 2000 Spectrophotometer.
Characterization of exosomes. The pellet from either BV2 cells or BV2-derived exosomes was lysated in PRO-PREP™ protein extraction solution (iNtRon Biotechnology, Seongnam, Korea), and the protein was loaded on 10% SDS polyacrylamide gel stained with Coomassie brilliant blue. Also, the protein was transferred to PVDF membranes for 2 h. Western blotting was confirmed with Alix (Cell signaling Technology, Beverly, MA, USA) antibody as an exosome marker. HRP-conjugated goat anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) antibody was used as a secondary antibody, and the immune-reactive band was visualized using a luminescent image analyzer LAS-1000 plus (Fuji Photo Film, Tokyo, Japan). Transmission electron microscopy was performed to examine the size and morphology of the exosomes. Exosomes (6.40 µg/µL) isolated from BV2 cells were prepared on a carbon-coating grid and negatively stained using 2% uranyl acetate for 1 min 45 . The prepared samples were observed using a JEOL 1200-EX II (Tokyo, Japan) equipped with an ES1000W Erlangshen CCD camera (Gatan, Pleasanton, CA, USA) at an accelerating voltage of 80 kV.
Confocal microscopy. BV2-derived exosomes were stained using PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. U87 cells, 2 × 10 5 , were incubated with either PKH26-labeled exosomes (red) or without exosomes as a negative control for 24 h at 37 °C in a 5% CO 2 incubator. The nuclei of U87 cells were stained with DAPI (blue), and U87 cells were fixed in 4% paraformaldehyde for 15 min at 4 °C before observing in Leica TCS SP8 STED CW confocal microscopy system (Leica Microsystems, Mannheim, Germany).

Real-time RT-PCR.
The cDNA was synthesized from miRNA with a Mir-X miRNA First Strand Synthesis and SYBR qRT-PCR Kit (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR with an iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) used primers and templates mixed with the SYBR Premix. DNA was amplified for 40 cycles of denaturation for 15 s at 95 °C and annealing for 60 s at 60 °C. The data generated from each PCR reaction were analyzed using the Data Analysis Module by iQ™5 optical system software (Bio-Rad). The relative changes in gene expression were calculated between selected target genes and ß-actin/ miRNAs (and U6) using the standard curve method. Primer sequences used are shown in Table 1.
Cell proliferation assay. U87  Altered microRNAs analysis. Expression profiling of mature exosomal miRNAs in microglial cells by the RH or ME49 strain of Toxoplasma infection was analyzed using various web tools. The miRNAs related to tumor development were identified using miRWalk2.0 (http:// mirwa lk. umm. uni-heide lberg. de) and filtered with more than 8.0 normalized signal intensity (log2). Among the miRNAs, differentially expressed miRNAs by greater than 1.5-fold changes were determined by filtering to analyze miRNAs altered by the RH or ME49 strain of Toxoplasma infection.

Data availability
The data used in the current study are available from the first author (BK Jung; mulddang@snu.ac.kr) upon reasonable request. And the microarray data of the current study are deposited in the GEO database under the accession number GPL32397.