Thank you for visiting Nature.com. The browser version you are using has limited CSS support. For the best experience, we recommend that you use an updated browser (or disable Compatibility Mode in Internet Explorer). In the meantime, to ensure continued support, we will render the site without styles and JavaScript.
Toxoplasma gondii is an intracellular protozoan parasite that modulates the microenvironment of the infected host and is known to be associated with the incidence of brain tumor growth. In this study, we hypothesize that exosomal miRNA-21 from Toxoplasma infection promotes brain tumor growth. Exosomes from Toxoplasma-infected BV2 microglia were characterized and internalization of U87 glioma cells was confirmed. Exosomal microRNA expression profiles were analyzed using arrays of microRNA and microRNA-21A-5p associated with Toxoplasma gondii and tumor sorting. We also investigated the mRNA levels of tumor-associated genes in U87 glioma cells by altering miR-21 levels in exosomes and the effect of exosomes on human U87 glioma cell proliferation. In exosomes of U87 glioma cells infected with Toxoplasma gondii, the expression of microRNA-21 is increased and the activity of antitumor genes (FoxO1, PTEN, and PDCD4) is reduced. BV2-derived exosomes infected with Toxoplasma induce proliferation of U87 glioma cells. Exosomes induce growth of U87 cells in a mouse tumor model. We suggest that increased exosomal miR-21 in Toxoplasma-infected BV2 microglia may play an important role as a cell growth promoter in U87 glioma cells by downregulating antitumor genes.
It is estimated that more than 18.1 million cases of advanced cancer were diagnosed worldwide in 2018, with about 297,000 central nervous system tumors diagnosed each year (1.6% of all tumors)1. Previous research has shown that risk factors for developing human brain tumors include various chemical products, family history, and ionizing radiation from head therapeutic and diagnostic equipment. However, the exact cause of these malignancies is unknown. Approximately 20% of all cancers worldwide are caused by infectious agents, including viruses, bacteria and parasites3,4. Infectious pathogens disrupt the host cell’s genetic mechanisms, such as DNA repair and the cell cycle, and can lead to chronic inflammation and damage to the immune system5.
Infectious agents associated with human cancer are the most common viral pathogens, including human papillomaviruses and hepatitis B and C viruses. Parasites can also play a potential role in the development of human cancer. Several parasite species, namely Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis and Hymenolepis nana, have been implicated in various types of human cancer 6,7,8.
Toxoplasma gondii is an intracellular protozoan that regulates the microenvironment of infected host cells. This parasite is estimated to infect approximately 30% of the world’s population, putting the entire population at risk9,10. Toxoplasma gondii can infect vital organs, including the central nervous system (CNS), and cause serious illnesses such as fatal meningitis and encephalitis, especially in immunocompromised patients9. However, Toxoplasma gondii can also alter the environment of the infected host by modulating cell growth and immune responses in immunocompetent individuals, leading to the maintenance of an asymptomatic chronic infection9,11. Interestingly, given the correlation between T. gondii prevalence and brain tumor incidence, some reports suggest that in vivo host environmental changes due to chronic T. gondii infection resemble the tumor microenvironment.
Exosomes are known as intercellular communicators that deliver biological content, including proteins and nucleic acids, from neighboring cells16,17. Exosomes can influence tumor-related biological processes such as anti-apoptosis, angiogenesis, and metastasis in the tumor microenvironment. In particular, miRNAs (miRNAs), small non-coding RNAs about 22 nucleotides in length, are important post-transcriptional gene regulators that control more than 30% of human mRNA through the miRNA-induced silencing complex (miRISC). Toxoplasma gondii can disrupt biological processes by controlling miRNA expression in infected hosts. Host miRNAs contain important signals for regulating host biological processes to achieve the parasite’s survival strategy. Thus, studying changes in the host miRNA profile upon infection with T. gondii can help us understand the interaction between the host and T. gondii more clearly. Indeed, Thirugnanam et al. 15 suggested that T. gondii promotes brain carcinogenesis by altering its expression on specific host miRNAs associated with tumor growth and found that T. gondii can cause gliomas in experimental animals.
This study focuses on the alteration of exosomal miR-21 in host microglia infected with Toxoplasma BV2. We observed a possible role of altered exosomal miR-21 in the growth of U87 glioma cells due to the retention in the nucleus of FoxO1/p27, which is the target of overexpressed miR-21.
Exosomes derived from BV2 were obtained using differential centrifugation and validated by various methods to prevent contamination with cellular components or other vesicles. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct patterns between proteins extracted from BV2 cells and exosomes (Figure 1A), and samples were assessed for the presence of Alix, which was analyzed by Western blotting of exosomal protein markers in . Alix labeling was found in exosome proteins but not in BV2 cell lysate proteins (Fig. 1B). In addition, purified RNA from exosomes derived from BV2 was analyzed using a bioanalyzer. 18S and 28S ribosomal subunits were rarely observed in the exosomal RNA migration pattern, indicating reliable purity (Figure 1C). Finally, transmission electron microscopy showed that the observed exosomes were about 60–150 nm in size and had a cup-like structure typical of exosome morphology (Fig. 1D).
Characterization of exosomes derived from BV2 cells. (A) Safety data sheet page. Proteins were isolated from BV2 cells or exosomes derived from BV2. Protein patterns differ between cells and exosomes. (B) Western blot analysis of an exosomal marker (Alix). (C) Evaluation of purified RNA from BV2 cells and BV2 derived exosomes using a bioanalyzer. Thus, 18S and 28S ribosomal subunits in BV2 cells were rarely found in exosomal RNA. (D) Transmission electron microscopy showed that exosomes isolated from BV2 cells were negatively stained with 2% uranyl acetate. Exosomes are approximately 60-150 nm in size and cup-shaped (Song and Jung, unpublished data).
Cellular internalization of BV2-derived exosomes into U87 human glioma cells was observed using confocal microscopy. PKH26 labeled exosomes are localized in the cytoplasm of U87 cells. Nuclei were stained with DAPI (Fig. 2A), indicating that BV2-derived exosomes can be internalized by host cells and influence the environment of recipient cells.
Internalization of BV2-derived exosomes into U87 glioma cells and BV2-derived exosomes infected with Toxoplasma RH induced proliferation of U87 glioma cells. (A) Exosomes engulfed by U87 cells measured by confocal microscopy. U87 glioma cells were incubated with exosomes labeled with PKH26 (red) or without control for 24 hours. The nuclei were stained with DAPI (blue) and then observed under a confocal microscope (scale bar: 10 μm, x 3000). (B) U87 glioma cell proliferation was determined by cell proliferation assay. U87 glioma cells were treated with exosomes for the indicated time. *P < 0.05 was obtained by Student’s t test. *P < 0.05 was obtained by Student’s t test. *P < 0,05 получено по t-критерию Стьюдента. *P < 0.05 by Student’s t-test. *P < 0.05 通过学生t 检验获得。 *P < 0.05 * P < 0,05, полученный с помощью t-критерия Стьюдента. * P < 0.05 obtained using Student’s t-test.
After confirming the internalization of BV2-derived exosomes into U87 glioma cells, we performed cell proliferation assays to investigate the role of BV2-derived Toxoplasma-derived exosomes in the development of human glioma cells. Treatment of U87 cells with exosomes from T. gondii-infected BV2 cells showed that T. gondii-infected BV2-derived exosomes caused significantly higher proliferation of U87 cells compared to control (Fig. 2B).
In addition, growth of U118 cells had the same results as U87, as Toxoplasma stimulated exosomes caused the highest levels of proliferation (data not shown). Based on these data, we can indicate that BV2-derived Toxoplasma-infected exosomes play an important role in glioma cell proliferation.
To investigate the effect of Toxoplasma-infected BV2-derived exosomes on tumor development, we injected U87 glioma cells into nude mice for a xenograft model and injected BV2-derived exosomes or RH-infected BV2-derived exosomes. After tumors became apparent after 1 week, each experimental group of 5 mice was divided according to tumor size to determine the same starting point, and tumor size was measured for 22 days.
In mice with the U87 xenograft model, significantly larger tumor size and weight were observed in the BV2-derived RH-infected exosome group at day 22 (Fig. 3A,B). On the other hand, there was no significant difference in tumor size between the BV2-derived exosome group and the control group after exosome treatment. In addition, mice injected with glioma cells and exosomes visually displayed the largest tumor volume in the group of RH-infected BV2-derived exosomes (Fig. 3C). These results demonstrate that BV2-derived Toxoplasma-infected exosomes induce glioma growth in a mouse tumor model.
Oncogenesis (AC) of BV2-derived exosomes in a U87 xenograft mouse model. Tumor size (A) and weight (B) were significantly increased in BALB/c nude mice treated with RH-infected exosomes derived from BV2. BALB/c nude mice (C) were injected subcutaneously with 1 x 107 U87 cells suspended in Matrigel mixture. Six days after injection, 100 μg of BV2-derived exosomes were treated in mice. Tumor size and weight were measured on the indicated days and after sacrifice, respectively. *P < 0.05. *P < 0.05. *Р < 0,05. *P < 0.05. *P < 0.05。 *P < 0.05。 *Р < 0,05. *P < 0.05.
The data showed that 37 miRNAs (16 overexpressed and 21 downexpressed) associated with immunity or tumor development were significantly altered in microglia after infection with the Toxoplasma RH strain (Fig. 4A). Relative expression levels of miR-21 among altered miRNAs were confirmed by real-time RT-PCR in exosomes derived from BV2, exosomes treated with BV2 and U87 cells. Expression of miR-21 showed a significant increase in exosomes from BV2 cells infected with Toxoplasma gondii (RH strain) (Fig. 4B). Relative expression levels of miR-21 in BV2 and U87 cells increased after uptake of altered exosomes (Fig. 4B). The relative levels of miR-21 expression in the brain tissues of tumor patients and mice infected with Toxoplasma gondii (ME49 strain) were higher than in controls, respectively (Fig. 4C). These results correlate with differences between the expression levels of predicted and confirmed microRNAs in vitro and in vivo.
Changes in the expression of exosomal miP-21a-5p in microglia infected with Toxoplasma gondii (RH). (A) Demonstrates significant changes in siRNA associated with immunity or tumor development following T. gondii RH infection. (B) Relative miR-21 expression levels were detected by real-time RT-PCR in BV2-derived exosomes, BV2-treated exosomes, and U87 cells. (C) Relative miR-21 expression levels were found in the brain tissues of tumor patients (N=3) and mice infected with Toxoplasma gondii (ME49 strain) (N=3). *P < 0.05 was obtained by Student’s t test. *P < 0.05 was obtained by Student’s t test. *P < 0,05 было получено с помощью t-критерия Стьюдента. *P < 0.05 was obtained using Student’s t-test. *P < 0.05 通过学生t 检验获得。 *P < 0.05 * P <0,05, полученный с помощью t-критерия Стьюдента. * P < 0.05 obtained using Student’s t-test.
Exosomes from RH-infected BV2 cells led to the growth of gliomas in vivo and in vitro (Fig. 2, 3). To detect relevant mRNAs, we examined mRNA levels of antitumor target genes, forkhead box O1 (FoxO1), PTEN, and programmed cell death 4 (PDCD4) in U87 cells infected with exosomes derived from BV2 or RH BV2. Bioinformatics analysis has shown that several tumor-associated genes, including the FoxO1, PTEN, and PDCD4 genes, have miR-2121,22 binding sites. mRNA levels of antitumor target genes were reduced in RH-infected BV2-derived exosomes compared to BV2-derived exosomes (Fig. 5A). FoxO1 showed reduced protein levels in RH-infected BV2-derived exosomes compared to BV2-derived exosomes (Figure 5B). Based on these results, we could confirm that exosomes derived from RH-infected BV2 downregulate anti-oncogenic genes, maintaining their role in tumor growth.
Toxoplasma RH-infected BV2-derived exosomes induce suppression of antitumor genes in U87 glioma cells by Toxoplasma RH-infected BV2-derived exosomes. (A) Real-time PCR of FoxO1, PTEN and PDCD4 expression in exosomes derived from T. gondii RH-infected BV2 compared to PBS exosomes. β-actin mRNA was used as a control. (B) FoxO1 expression was determined by Western blotting and densitometry data were statistically evaluated using the ImageJ program. *P < 0.05 was obtained by Student’s t test. *P < 0.05 was obtained by Student’s t test. *P < 0,05 было получено с помощью t-критерия Стьюдента. *P < 0.05 was obtained using Student’s t-test. *P < 0.05 通过学生t 检验获得。 *P < 0.05 * P <0,05, полученный с помощью t-критерия Стьюдента. * P < 0.05 obtained using Student’s t-test.
To understand the effect of miP-21 in exosomes on tumor-associated gene regulation, U87 cells were transfected with an inhibitor of miP-21 using Lipofectamine 2000 and the cells were harvested 24 hours after transfection. FoxO1 and p27 expression levels in cells transfected with miR-21 inhibitors were compared to cells treated with BV2-derived exosomes using qRT-PCR (Fig. 6A,B). Transfection of the miR-21 inhibitor into U87 cells significantly downregulated FoxO1 and p27 expression (FIG. 6).
RH-infected exosomal BV2-derived miP-21 altered FoxO1/p27 expression in U87 glioma cells. U87 cells were transfected with miP-21 inhibitor using Lipofectamine 2000 and cells were harvested 24 hours after transfection. FoxO1 and p27 expression levels in cells transfected with miR-21 inhibitors were compared to levels in cells treated with BV2-derived exosomes using qRT-PCR (A, B).
To escape the host’s immune response, the Toxoplasma parasite transforms into a tissue cyst. They parasitize various tissues, including the brain, heart, and skeletal muscle, throughout the lifetime of the host and modulate the host’s immune response. In addition, they can regulate the cell cycle and apoptosis of host cells, promoting their proliferation14,24. Toxoplasma gondii predominantly infects host dendritic cells, neutrophils, and monocyte/macrophage lineage, including brain microglia. Toxoplasma gondii induces the differentiation of macrophages of the M2 phenotype, affects wound healing after pathogen infection, and is also associated with hypervascularization and granulomatous fibrosis. This behavioral pathogenesis of Toxoplasma infection may be related to markers associated with tumor development. The hostile environment regulated by Toxoplasma may resemble the corresponding precancer. Therefore, it can be assumed that Toxoplasma infection should contribute to the development of brain tumors. In fact, high rates of Toxoplasma infection have been reported in the serum of patients with various brain tumors. In addition, Toxoplasma gondii may be another carcinogenic effector and act synergistically to help other infectious carcinogens develop brain tumors. In this regard, it is worth noting that P. falciparum and Epstein-Barr virus synergistically contribute to the formation of Burkitt’s lymphoma.
The role of exosomes as regulators in the field of cancer research has been extensively investigated. However, the role of exosomes between parasites and infected hosts remains poorly understood. So far, various regulators, including secreted proteins, have explained the biological processes by which protozoan parasites resist host attack and perpetuate infection. Recently, there has been a growing concept that protozoan-associated microvesicles and their microRNAs interact with host cells to create a favorable environment for their survival. Therefore, further studies are needed to discover the relationship between altered exosomal miRNAs and glioma cell proliferation. MicroRNA alteration (cluster genes miR-30c-1, miR-125b-2, miR-23b-27b-24-1 and miR-17-92) binds to the STAT3 promoter in toxoplasma-infected human macrophages, is regulated and induces anti-apoptosis in response to Toxoplasma gondii infection 29 . Toxoplasma infection increases the expression of miR-17-5p and miR-106b-5p, which are associated with several hyperproliferative diseases 30 . These data suggest that host miRNAs regulated by Toxoplasma infection are important molecules for parasite survival and pathogenesis in host biological behavior.
Altered miRNAs can influence various types of behavior during the initiation and progression of malignant cells, including gliomas: self-sufficiency of growth signals, insensitivity to growth-inhibiting signals, apoptosis evasion, unlimited replicative potential, angiogenesis, invasion and metastasis, and inflammation. In glioma, altered miRNAs have been identified in several expression profiling studies.
In the present study, we confirmed high levels of miRNA-21 expression in toxoplasma-infected host cells. miR-21 has been identified as one of the most frequently overexpressed microRNAs in solid tumors, including gliomas, 33 and its expression correlates with the grade of glioma. Accumulating evidence suggests that miR-21 is a novel oncogene that acts as an anti-apoptotic factor in glioma growth and is highly overexpressed in tissues and plasma of human brain malignancies. Interestingly, miR-21 inactivation in glioma cells and tissues triggers the inhibition of cell proliferation due to caspase-dependent apoptosis. Bioinformatic analysis of miR-21 predicted targets revealed multiple tumor suppressor genes associated with apoptosis pathways, including programmed cell death 4 (PDCD4), tropomyosin (TPM1), PTEN, and forkhead box O1 (FoxO1), with the miR-2121 binding site. .22.38.
FoxO1, as one of the transcription factors (FoxO), is involved in the development of various types of human cancer and can regulate the expression of tumor suppressor genes such as p21, p27, Bim, and FasL40. FoxO1 can bind and activate cell cycle inhibitors such as p27 to suppress cell growth. Moreover, FoxO1 is a key effector of PI3K/Akt signaling and regulates many biological processes such as cell cycle progression and cell differentiation through activation of p2742 transcription.
In conclusion, we believe that exosomal miR-21 derived from Toxoplasma-infected microglia may play an important role as a growth regulator of glioma cells (Fig. 7). However, further studies are needed to find a direct link between exosomal miR-21, altered Toxoplasma infection, and glioma growth. These results are expected to provide a starting point for studying the relationship between Toxoplasma infection and the incidence of glioma.
A schematic diagram of the mechanism of glioma (brain) carcinogenesis is proposed in this study. The author draws in PowerPoint 2019 (Microsoft, Redmond, WA).
All experimental protocols in this study, including the use of animals, were in accordance with the Seoul National University Animal Care and User Committee Standard Ethical Guidelines and were approved by the Institutional Review Board of the Seoul National University School of Medicine (IRB number SNU-150715). -2). All experimental procedures were carried out in accordance with ARRIVE recommendations.
BV2 mouse microglia and U87 human glioma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene, Seoul, Korea) and Roswell Park Memorial Institute’s Medium (RPMI; Welgene), respectively, each containing 10% fetal bovine serum, 4 mM l-glutamine, 0.2 mM penicillin and 0.05 mM streptomycin. Cells were cultured in an incubator with 5% CO2 at 37°C. Another glioma cell line, U118, was used for comparison with U87 cells.
To isolate exosomes from T. gondii-infected RH and ME49 strains, T. gondii tachyzoites (RH strain) were harvested from the abdominal cavity of 6-week-old BALB/c mice injected 3-4 days prior. Tachyzoites were washed three times with PBS and purified by centrifugation in 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43. To obtain tachyzoites of strain ME49, BALB/c mice were intraperitoneally injected with 20 tissue cysts and tachyzoite transformation in cysts was collected by washing the abdominal cavity on the 6-8th day after infection (PI). Mice infected with PBS. ME49 tachyzoites were grown in cells supplemented with 100 μg/ml penicillin (Gibco/BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Gibco/BRL), and 5% fetal bovine serum (Lonza, Walkersville, MD ). ., USA) at 37 °C and 5% carbon dioxide. After cultivation in Vero cells, ME49 tachyzoites were passed twice through a 25 gauge needle and then through a 5 µm filter to remove debris and cells. After washing, the tachyzoites were resuspended in PBS44. Tissue cysts of Toxoplasma gondii strain ME49 were maintained by intraperitoneal injection of cysts isolated from the brain of infected C57BL/6 mice (Orient Bio Animal Center, Seongnam, Korea). The brains of ME49-infected mice were harvested after 3 months of PI and minced under a microscope to isolate cysts. The infected mice were kept under special pathogen-free conditions (SPF) at the Seoul National University School of Medicine.
Total RNA was extracted from BV2-derived exosomes, BV2 cells and tissues using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, including the incubation time for the elution step. The RNA concentration was determined on a NanoDrop 2000 spectrophotometer. The quality of RNA microarrays was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands).
DMEM with 10% exosome-poor FBS was prepared by ultracentrifugation at 100,000g for 16 hours at 4°C and filtered through a 0.22 µm filter (Nalgene, Rochester, NY, USA). BV2 cells, 5 × 105, were cultured in DMEM containing 10% exosome-depleted FBS and 1% antibiotics at 37°C and 5% CO2. After 24 hours of incubation, tachyzoites of strain RH or ME49 (MOI = 10) were added to the cells and non-invading parasites were removed within an hour and refilled with DMEM. Exosomes from BV2 cells were isolated by modified differential centrifugation, the most widely used method. Resuspend the exosome pellet in 300 µl PBS for RNA or protein analysis. The concentration of isolated exosomes was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) and a NanoDrop 2000 spectrophotometer.
Precipitates from BV2 cells or exosomes derived from BV2 were lysed in PRO-PREP™ protein extraction solution (iNtRon Biotechnology, Seongnam, Korea) and proteins were loaded onto Coomassie brilliant blue stained 10% SDS polyacrylamide gels. In addition, proteins were transferred to PVDF membranes for 2 hours. Western blots were validated using the Alix antibody (Cell Signaling Technology, Beverly, MA, USA) as an exosomal marker. HRP-conjugated goat anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) and a LAS-1000 plus luminescent image analyzer (Fuji Photographic Film, Tokyo, Japan) were used as a secondary antibody. . Transmission electron microscopy was performed to study the size and morphology of exosomes. Exosomes isolated from BV2 cells (6.40 µg/µl) were prepared on carbon-coated meshes and negatively stained with 2% uranyl acetate for 1 min. The prepared samples were observed at an accelerating voltage of 80 kV using a JEOL 1200-EX II (Tokyo, Japan) equipped with an ES1000W Erlangshen CCD camera (Gatan, Pleasanton, CA, USA).
BV2-derived exosomes were stained using the PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes at room temperature. U87 cells, 2×105, with PKH26-labeled exosomes (red) or no exosomes as a negative control, were incubated at 37°C for 24 hours in a 5% CO2 incubator. U87 cell nuclei were stained with DAPI (blue), U87 cells were fixed in 4% paraformaldehyde for 15 min at 4°C and then analyzed in a Leica TCS SP8 STED CW confocal microscope system (Leica Microsystems, Mannheim, Germany). observable.
cDNA was synthesized from siRNA using Mir-X siRNA first strand synthesis and SYBR qRT-PCR kit (Takara Bio Inc., Shiga, Japan). Real time quantitative PCR was performed using the iQ5 real time PCR detection system (Bio-Rad, Hercules, CA, USA) using primers and templates mixed with SYBR Premix. DNA was amplified for 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 60 s. The data from each PCR reaction was analyzed using the data analysis module of the iQ™5 optical system software (Bio-Rad). Relative changes in gene expression between selected target genes and β-actin/siRNA (and U6) were calculated using the standard curve method. The primer sequences used are shown in Table 1.
3 x 104 U87 glioma cells were seeded in 96-well plates and mixed with Toxoplasma-infected exosomes derived from BV2 (50 μg/mL) or nonpulse exosomes derived from BV2 (50 μg/mL) as controls at 12, 18 and 36 hours. The cell proliferation rate was determined using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) (Supplementary Figures S1-S3) 46 .
5-week-old female BALB/c nude mice were purchased from Orient Bio (Seongnam-si, South Korea) and kept individually in sterile cages at room temperature (22±2°C) and humidity (45±15°C). %) at room temperature (22±2°C) and humidity (45±15%). A 12-hour light cycle and a 12-hour dark cycle were performed under SPF (Seoul National University School of Medicine Animal Center). Mice were randomly divided into three groups of 5 mice each and all groups were injected subcutaneously with 400 ml of PBS containing 1 x 107 U87 glioma cells and growth factor reduced BD Matrigel™ (BD Science, Miami, FL, USA). Six days after tumor injection, 200 mg of exosomes derived from BV2 cells (with/without Toxoplasma infection) were injected into the tumor site. Twenty-two days after tumor infection, the tumor size of mice in each group was measured with a caliper three times a week, and the tumor volume was calculated by the formula: 0.5×(width)×2×length.
MicroRNA expression analysis using miRCURYTM LNA miRNA array, 7th generation has, mmu and rno arrays (EXIQON, Vedbaek, Denmark) covering 1119 well-characterized mice among 3100 human, mouse and rat miRNA capture probes. During this procedure, 250 to 1000 ng of total RNA was removed from the 5′-phosphate by treatment with calf intestinal alkaline phosphatase followed by labeling with Hy3 green fluorescent dye. The labeled samples were then hybridized by loading microarray slides using a hybridization chamber kit (Agilent Technologies, Santa Clara, CA, USA) and a hybridization slide kit (Agilent Technologies). Hybridization was carried out for 16 hours at 56°C, then the microarrays were washed in accordance with the manufacturer’s recommendations. The processed microarray slides were then scanned using an Agilent G2565CA microarray scanner system (Agilent Technologies). Scanned images were imported using Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies) and the fluorescence intensity of each image was quantified using the corresponding GAL file of the modified Exiqon protocol. Microarray data for the current study are deposited in the GEO database under accession number GPL32397.
Expression profiles of mature exosomal miRNAs in microglia of RH or ME49 strains infected with Toxoplasma were analyzed using various network tools. miRNAs associated with tumor development were identified using miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) and filtered out with normalized signal intensity (log2) greater than 8.0. Among miRNAs, differentially expressed miRNAs were found to be more than 1.5-fold altered by filter analysis of miRNAs altered by RH or ME49 strains infected with T. gondii.
Cells were seeded in six-well plates (3 x 105 cells/well) in opti-MEM (Gibco, Carlsbad, CA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The transfected cells were cultured for 6 hours and then the medium was changed to fresh complete medium. Cells were harvested 24 hours after transfection.
Statistical analysis was mainly performed using Student’s t-test with Excel software (Microsoft, Washington, DC, USA). For experimental animal analysis, a two-way ANOVA was performed using Prism 3.0 software (GraphPad Software, La Jolla, CA, USA). P-values < 0.05 were regarded as statistically significant. P-values ​​< 0.05 were regarded as statistically significant. Значения P <0,05 считались статистически значимыми. P values ​​<0.05 were considered statistically significant. P 值< 0.05 被认为具有统计学意义。 P 值< 0.05 Значения P <0,05 считались статистически значимыми. P values ​​<0.05 were considered statistically significant.
All experimental protocols used in this study were approved by the Institutional Review Board of the Seoul National University School of Medicine (IRB number SNU-150715-2).
The data used in this study are available upon reasonable request from the first author (BK Jung; mulddang@snu.ac.kr). And the microarray data for the current study is deposited in the GEO database under registration number GPL32397.
Furley, J. et al. Estimated global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Interpretation. J. Ruck 144, 1941–1953 (2019).
Rasheed, S., Rehman, K. & Akash, MS An insight into the risk factors of brain tumors and their therapeutic interventions. Rasheed, S., Rehman, K. & Akash, MS An insight into the risk factors of brain tumors and their therapeutic interventions. Rashid, S., Rehman, K. and Akash, M.S. A review of risk factors for brain tumors and major therapeutic interventions. Rasheed, S., Rehman, K. & Akash, MS 深入了解脑肿瘤的危险因素及其治疗干预措施。 Rasheed, S., Rehman, K. & Akash, MS Deep understanding of brain tumor risk factors and therapeutic interventions. Rashid, S., Rehman, K. and Akash, M.S. A review of risk factors for brain tumors and major therapeutic interventions. Biomedical science. Pharmacist. 143, 112119 (2021).
Kato, I., Zhang, J. & Sun, J. Bacterial-viral interactions in human orodigestive and female genital tract cancers: A summary of epidemiologic and laboratory evidence. Kato, I., Zhang, J. & Sun, J. Bacterial-viral interactions in human orodigestive and female genital tract cancers: A summary of epidemiologic and laboratory evidence. Kato I., Zhang J. and Sun J. Bacterial-viral interactions in cancer of the human gastrointestinal tract and female genital tract: a summary of epidemiological and laboratory data. Kato, I., Zhang, J. & Sun, J. 人类口腔消化道癌和女性生殖道癌中的细菌-病毒相互作用：流行病学和实验室证据总结。 Kato, I., Zhang, J. & Sun, J. Bacterio-viral interaction in human oral cavity digestion and female reproductive tract: summary of popular disease science and laboratory evidence. Kato I., Zhang J. and Sun J. Bacterial-viral interactions in human gastrointestinal cancer and female genital cancer: a summary of epidemiological and laboratory data. Cancer 14, 425 (2022).
Magon, KL & Parish, JL From infection to cancer: How DNA tumour viruses alter host cell central carbon and lipid metabolism. Magon, KL & Parish, JL From infection to cancer: How DNA tumor viruses alter host cell central carbon and lipid metabolism. Mahon, K.L. and Parish, J.L. Fire infection to cancer: how DNA-based tumor viruses alter host cell central carbon and lipid metabolism. Magon, KL & Parish, JL 从感染到癌症：DNA 肿瘤病毒如何改变宿主细胞的中心碳和脂质代谢。 Magon, KL & Parish, JL From infection to cancer: how DNA tumor viruses alter host cell central carbon and lipid metabolism. Mahon, K.L. and Parish, J.L. Fires infection to cancer: how DNA tumor viruses alter central carbon and lipid metabolism in host cells. Open Biology. 11, 210004 (2021).
Correia da Costa, J.M. et al. Catechol estrogens of schistosomes and liver flukes and helminth-associated cancer. front. hot inside. 5, 444 (2014).