ARPC1B promotes mesenchymal phenotype maintenance and radiotherapy resistance by blocking TRIM21-mediated degradation of IFI16 and HuR in glioma stem cells.

BACKGROUND: Intratumoral heterogeneity is the primary challenge in the treatment of glioblastoma (GBM). The presence of glioma stem cells (GSCs) and their conversion between different molecular phenotypes contribute to the complexity of heterogeneity, culminating in preferential resistance to radiotherapy. ARP2/3 (actin-related protein-2/3) complexes (ARPs) are associated with cancer migration, invasion and differentiation, while the implications of ARPs in the phenotype and resistance to radiotherapy of GSCs remain unclear. METHODS: We screened the expression of ARPs in TCGA-GBM and CGGA-GBM databases. Tumor sphere formation assays and limiting dilution assays were applied to assess the implications of ARPC1B in tumorigenesis. Apoptosis, comet, γ-H2AX immunofluorescence (IF), and cell cycle distribution assays were used to evaluate the effect of ARPC1B on radiotherapy resistance. Immunoprecipitation (IP) and mass spectrometry analysis were used to detect ARPC1B-interacting proteins. Immune blot assays were performed to evaluate protein ubiquitination, and deletion mutant constructs were designed to determine the binding sites of protein interactions. The Spearman correlation algorithm was performed to screen for drugs that indicated cell sensitivity by the expression of ARPC1B. An intracranial xenograft GSC mouse model was used to investigate the role of ARPC1B in vivo. RESULTS: We concluded that ARPC1B was significantly upregulated in MES-GBM/GSCs and was correlated with a poor prognosis. Both in vitro and in vivo assays indicated that knockdown of ARPC1B in MES-GSCs reduced tumorigenicity and resistance to IR treatment, whereas overexpression of ARPC1B in PN-GSCs exhibited the opposite effects. Mechanistically, ARPC1B interacted with IFI16 and HuR to maintain protein stability. In detail, the Pyrin of IFI16 and RRM2 of HuR were implicated in binding to ARPC1B, which counteracted TRIM21-mediated degradation of ubiquitination to IFI16 and HuR. Additionally, the function of ARPC1B was dependent on IFI16-induced activation of NF-κB pathway and HuR-induced activation of STAT3 pathway. Finally, we screened AZD6738, an ataxia telangiectasia mutated and rad3-related (ATR) inhibitor, based on the expression of ARPC1B. In addition to ARPC1B expression reflecting cellular sensitivity to AZD6738, the combination of AZD6738 and radiotherapy exhibited potent antitumor effects both in vitro and in vivo. CONCLUSION: ARPC1B promoted MES phenotype maintenance and radiotherapy resistance by inhibiting TRIM21-mediated degradation of IFI16 and HuR, thereby activating the NF-κB and STAT3 signaling pathways, respectively. AZD6738, identified based on ARPC1B expression, exhibited excellent anti-GSC activity in combination with radiotherapy.

Exosomal miR-1246 from glioma patient body fluids drives the differentiation and activation of myeloid-derived suppressor cells.

Glioma is a heterogeneous cellular environment in which immune cells play critical roles in tumor progression. Myeloid-derived suppressor cells (MDSCs) contribute to the formation of the immunosuppressive microenvironment of glioma; however, how glioma cells interact with MDSCs and how this interaction affects the function of other immune cells are unclear. Glioma cells can systemically communicate with immune cells via the secretion of exosomes, which contain microRNAs (miRNAs). Leveraging miRNA sequencing of exosomes, we identified enrichment of miR-1246 in glioma-derived exosomes and exosomes isolated from the cerebrospinal fluid (CSF) of glioma patients. We demonstrated that miR-1246 drives the differentiation and activation of MDSCs in a dual specificity phosphatase 3 (DUSP3)/extracellular signal­regulated kinase (ERK)-dependent manner. In addition, postoperative CSF exosomal miR-1246 expression was found to be associated with the glioma recurrence rate. Hypoxia, a well-recognized feature of the glioblastoma microenvironment, increased miR-1246 levels in glioma-derived exosomes by enhancing miR-1246 transcription and selective packaging via upregulation of POU class 5 homeobox 1 (POU5F1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Importantly, we identified a mechanism of 2-methoxyestradiol, a microtubule inhibitor currently undergoing clinical trials for glioblastoma. 2-Methoxyestradiol suppresses MDSC activation by inhibiting hypoxia-driven exosomal miR-1246 expression in glioma cells and PD-L1 expression in MDSCs.

Hypoxic glioma-derived exosomes promote M2-like macrophage polarization by enhancing autophagy induction.

Exosomes participate in intercellular communication and glioma microenvironment modulation, but the exact mechanisms by which glioma-derived exosomes (GDEs) promote the generation of the immunosuppressive microenvironment are still unclear. Here, we investigated the effects of GDEs on autophagy, the polarization of tumor-associated macrophages (TAMs), and glioma progression. Compared with normoxic glioma-derived exosomes (N-GDEs), hypoxic glioma-derived exosomes (H-GDEs) markedly facilitated autophagy and M2-like macrophage polarization, which subsequently promoted glioma proliferation and migration in vitro and in vivo. Western blot and qRT-PCR analyses indicated that interleukin 6 (IL-6) and miR-155-3p were highly expressed in H-GDEs. Further experiments showed that IL-6 and miR-155-3p induced M2-like macrophage polarization via the IL-6-pSTAT3-miR-155-3p-autophagy-pSTAT3 positive feedback loop, which promotes glioma progression. Our study clarifies a mechanism by which hypoxia and glioma influence autophagy and M2-like macrophage polarization via exosomes, which could advance the formation of the immunosuppressive microenvironment. Our findings suggest that IL-6 and miR-155-3p may be novel biomarkers for diagnosing glioma and that treatments targeting autophagy and the STAT3 pathway may contribute to antitumor immunotherapy.

MicroRNA-29a-3p delivery via exosomes derived from engineered human mesenchymal stem cells exerts tumour suppressive effects by inhibiting migration and vasculogenic mimicry in glioma.

Vasculogenic mimicry (VM), the formation of an alternative microvascular circulation independent of VEGF-driven angiogenesis, is reluctant to anti-angiogenesis therapy for glioma patients. However, treatments targeting VM are lacking due to the poor understanding of the molecular mechanism involved in VM formation. By analysing the TCGA database, microRNA-29a-3p (miR-29a-3p) was found to be highly expressed in normal brain tissue compared with glioma. An in vitro study revealed an inhibitory role for miR-29a-3p in glioma cell migration and VM formation, and further study confirmed that ROBO1 is a direct target of miR-29a-3p. Based on this, we engineered human mesenchymal stem cells (MSCs) to produce miR-29a-3p-overexpressing exosomes. Treatment with these exosomes attenuated migration and VM formation in glioma cells. Moreover, the anti-glioma role of miR-29a-3p and miR-29a-3p-overexpressing exosomes were confirmed in vivo. Overall, the present study demonstrates that MSCs can be used to produce miR-29a-3p-overexpressing exosomes, which have great potential for anti-VM therapy and may act as supplements to anti-angiogenetic therapy in the clinic.

Exosomes derived from hypoxic glioma deliver miR-1246 and miR-10b-5p to normoxic glioma cells to promote migration and invasion.

Hypoxia is an important feature of the tumor microenvironment and is associated with glioma progression and patient outcome. Exosomes have been implicated in the intercellular communication in the tumor microenvironment. However, the effects of hypoxic glioma exosomes on glioma migration and invasion and the underlying mechanisms remain poorly understood. In this study, we found that exosomes derived from hypoxic glioma cells (H-GDEs) promoted normoxic glioma migration and invasion in vitro and in vivo. Given that exosomes can regulate recipient cell functions by delivering microRNAs, we further revealed miR-1246 and miR-10b-5p were upregulated significantly in H-GDEs and delivered to normoxic glioma cells by H-GDEs. Moreover, we determined the clinical relevance of miR-1246 and miR-10b-5p in glioma patients. Subsequent investigations indicated that miR-1246 and miR-10b-5p markedly induced glioma migration and invasion in vitro and in vivo. Finally, we demonstrated that miR-1246 and miR-10b-5p induced glioma migration and invasion by directly targeting FRK and TFAP2A respectively. In conclusion, our findings suggest that the hypoxic microenvironment stimulates glioma to generate miR-1246- and miR-10b-5p-rich exosomes that are delivered to normoxic glioma cells to promote their migration and invasion; treatment targeting miR-1246 and miR-10b-5p may impair the motility of gliomas, providing a novel direction for the development of antitumor therapy.

Transfer of MicroRNA via Macrophage-Derived Extracellular Vesicles Promotes Proneural-to-Mesenchymal Transition in Glioma Stem Cells.

Proneural-to-mesenchymal transition (PMT) is a common process in glioblastoma (GBM) progression that leads to increased radiotherapy resistance. However, the mechanism underlying PMT is poorly understood. Here, we found that tumor-associated macrophages triggered PMT in glioma stem cells (GSC) via small extracellular vesicles (sEV). sEVs from monocyte-derived macrophages transferred miR-27a-3p, miR-22-3p, and miR-221-3p to GSCs, and these miRNAs promoted several mesenchymal phenotypes in proneural (PN) GSCs by simultaneously targeting CHD7 We found that CHD7 played a critical role in the maintenance of the PN phenotype, and CHD7 knockdown significantly promoted PMT in GSCs via the RelB/P50 and p-STAT3 pathways. The induction of PMT by sEVs containing miR-27a-3p, miR-22-3p, and miR-221-3p in a xenograft nude mouse model exacerbated radiotherapy resistance and thus decreased the benefits of radiotherapy. Collectively, these findings identified macrophage-derived sEVs as key regulators of PMT in GSCs and demonstrated that CHD7 is a novel inhibitor of PMT.

mTORC2/Rac1 Pathway Predisposes Cancer Aggressiveness in IDH1-Mutated Glioma.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in lower grade gliomas. The neomorphic enzyme activity of IDH mutants leads to tumor formation through epigenetic alteration, dysfunction of dioxygenases, and metabolic reprogramming. However, it remains elusive as to how IDH mutants regulate the pathways associated with oncogenic transformation and aggressiveness. In the present study, by using unbiased transcriptomic profiling, we showed that IDH1 mutations result in substantial changes in the gene sets that govern cellular motility, chemotaxis, and invasion. Mechanistically, rapamycin-insensitive companion of mammalian target of rapamycin (Rictor)/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling plays an essential role in the motility and proliferation of IDH1-mutated cells by prompting cytoskeleton reorganization, lamellipodia formation, and enhanced endocytosis. Targeting the Rictor/Rac1 pathway suppresses IDH1-mutated cells by limiting endocytosis and cell proliferation. Overall, our findings indicate a novel metabolic reprogramming mechanism of IDH1-mutated cells by exploiting metabolites from the extracellular milieu. Targeting the Rictor/Rac1 pathway could be an alternative therapeutic strategy for IDH1-mutated malignancies.

Cullin-7 (CUL7) is overexpressed in glioma cells and promotes tumorigenesis via NF-κB activation.

BACKGROUND: Cullin-7 (CUL7) is a member of the DOC domain-containing cullin family and is involved in the regulation of cell transformation. However, the clinical significance, potential mechanism and upstream regulators of CUL7 in malignant gliomas remain to be determined. METHODS: Expression level data and clinical information were obtained via the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, immunohistochemistry (IHC) and western blot analysis. Gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of CUL7. RNA silencing was performed using siRNA or lentiviral constructs in U87MG and U251 glioma cell lines and GSC267 glioma stem cells. CUL7 overexpression was performed using the GV141-CUL7 plasmid construct. In addition, overexpression of miR-3940-5p was performed and validated by quantitative real-time PCR (qRT-PCR). Cells were characterized in vitro or in vivo to evaluate their molecular status, cell proliferation, invasion, and migration by Cell Counting Kit (CCK)-8, EdU, flow cytometry, colony formation, Transwell and 3D tumour spheroid invasion assays. Coimmunoprecipitation (co-IP) and western blotting were performed to test the mechanisms of activation of the NF-κB signalling pathway. RESULTS: High CUL7 expression was associated with a high tumour grade, a mesenchymal molecular glioma subtype and a poor prognosis in patients. Gene silencing of CUL7 in U87MG and U251 cells significantly inhibited tumour growth, invasion and migration in vitro and in vivo. Western blot analysis revealed that cyclin-dependent kinase inhibitors and epithelial-mesenchymal transition (EMT) molecular markers changed under CUL7 silencing conditions. In contrast, CUL7 overexpression promoted tumour growth, invasion and migration. Gene set enrichment analysis (GSEA) and western blot analysis revealed that CUL7 was positively associated with the NF-κB pathway. Moreover, with coimmunoprecipitation assays, we discovered that CUL7 physically associated with MST1, which further led to ubiquitin-mediated MST1 protein degradation, which promoted activation of the NF-κB signalling pathway. Finally, CUL7 was found to be downregulated by miR-3940-5p, which suppressed the development of gliomas. CONCLUSIONS: Our findings indicate that CUL7 plays a significant role in promoting tumorigenesis via NF-κB activation and that it can be negatively regulated by miR-3940-5p in human gliomas. Furthermore, CUL7 might be a candidate molecular target for the treatment of glioma.

IDH mutation in glioma: molecular mechanisms and potential therapeutic targets.

Isocitrate dehydrogenase (IDH) enzymes catalyse the oxidative decarboxylation of isocitrate and therefore play key roles in the Krebs cycle and cellular homoeostasis. Major advances in cancer genetics over the past decade have revealed that the genes encoding IDHs are frequently mutated in a variety of human malignancies, including gliomas, acute myeloid leukaemia, cholangiocarcinoma, chondrosarcoma and thyroid carcinoma. A series of seminal studies further elucidated the biological impact of the IDH mutation and uncovered the potential role of IDH mutants in oncogenesis. Notably, the neomorphic activity of the IDH mutants establishes distinctive patterns in cancer metabolism, epigenetic shift and therapy resistance. Novel molecular targeting approaches have been developed to improve the efficacy of therapeutics against IDH-mutated cancers. Here we provide an overview of the latest findings in IDH-mutated human malignancies, with a focus on glioma, discussing unique biological signatures and proceedings in translational research.

Hypoxia-induced lncRNA PDIA3P1 promotes mesenchymal transition via sponging of miR-124-3p in glioma.

Hypoxia is a critical factor in the malignant progression of glioma, especially for the highly-invasive mesenchymal (MES) subtype. But the detailed mechanisms in hypoxia-induced glioma MES transition remain elusive. Pseudogenes, once considered to be non-functional relics of evolution, are emerging as a critical factor in human tumorigenesis and progression. Here, we investigated the clinical significance, biological function, and mechanisms of protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) in hypoxia-induced glioma MES transition. In this study, we found that PDIA3P1 expression was closely related to tumor degree, transcriptome subtype, and prognosis in glioma patients. Enrichment analysis found that high PDIA3P1 expression was associated with epithelial-mesenchymal transition, extracellular matrix (ECM) disassembly, and angiogenesis. In vitro study revealed that overexpression of PDIA3P1 enhanced the migration and invasion capacity of glioma cells, while knockdown of PDIA3P1 induced the opposite effect. Further studies revealed that PDIA3P1 functions as a ceRNA, sponging miR-124-3p to modulate RELA expression and activate the downstream NF-κB pathway, thus promoting the MES transition of glioma cells. In addition, Hypoxia Inducible Factor 1 was confirmed to directly bind to the PDIA3P1 promotor region and activate its transcription. In conclusion, PDIA3P1 is a crucial link between hypoxia and glioma MES transition through the PDIA3P1-miR-124-3p-RELA axis, which may serve as a prognostic indicator and potential therapeutic target for glioma treatment.

Nitidine Chloride Is a Potential Alternative Therapy for Glioma Through Inducing Endoplasmic Reticulum Stress and Alleviating Epithelial-Mesenchymal Transition.

Background: Malignant glioma is a lethal brain tumor that is highly resistant to standard therapy. Our research aims to explore the suppressive effects of nitidine chloride (NC) on gliomas and the mechanisms involved, showing that it is a potential agent for integrative therapy of gliomas. Methods: After glioma cells were treated with NC, several experiments were performed to evaluate NC's antitumor effects. CCK-8 assay was used to detect viability. Transwell and 3-dimensional spheroid invasion assays were used to evaluate motility of glioma in vitro, and the sphere-formation assay showed NC's influence on glioma stem cells. Apoptosis and intracellular reactive oxygen species were measured by means of flow cytometry. Subcellular structures were observed through transmission electron microscopy. Western blot analysis reflected expression of endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) marker proteins. An orthotopic xenograft model was established to investigate the tumor suppressive effects in vivo. Results: Nitidine chloride inhibited glioma cell migration and invasion in vitro, downregulated the EMT proteins, and suppressed sphere formation of glioma stem cells. Furthermore, NC induced persistent ER stress that contributed to apoptosis and reactive oxygen species production. The xenograft model showed that NC effectively restricted glioma growth and invasion in vivo. Furthermore, we confirmed the signaling pathways that ER stress downregulates C/EBPß and slug, as well as inhibition of the AKT/GSK3ß/ß-catenin axis caused by NC, in U-87 MG. Conclusion: We demonstrated that NC inhibits gliomas in vitro and in vivo by activating ER stress and downregulating EMT, which provides a basis for glioma therapy.

Hypoxic glioma-derived exosomes deliver microRNA-1246 to induce M2 macrophage polarization by targeting TERF2IP via the STAT3 and NF-κB pathways.

Exosomes are emerging as important elements that participate in intercellular communication and tumor microenvironment modulation, but the exact mechanisms by which tumor exosomes facilitate the generation of the immunosuppressive microenvironment remain unclear. Here we investigated the effects of glioma-derived exosomes (GDEs) on macrophage polarization and glioma progression. We also performed microRNA sequencing analysis of GDEs to identify the microRNA that mediated macrophage polarization. The microRNA-associated intracellular signaling pathway in macrophages was further investigated. Compared with normoxic glioma-derived exosomes (N-GDEs), hypoxic glioma-derived exosomes (H-GDEs) markedly induced M2 macrophage polarization, which subsequently promoted glioma proliferation, migration and invasion in vitro and in vivo. MicroRNA sequencing analysis identified miR-1246 as the most enriched microRNA in H-GDEs. Moreover, miR-1246 was enriched in the CSF of GBM patients and decreased after tumor resection. Further investigation determined that miR-1246 mediated H-GDE-induced M2 macrophage polarization by targeting TERF2IP to activate the STAT3 signaling pathway and inhibit the NF-κB signaling pathway. Our study elucidated a mechanism by which hypoxia and glioma influence M2 macrophage polarization via exosomes, which could facilitate the formation of the immunosuppressive microenvironment. Moreover, our results suggested that miR-1246 in the CSF of GBM patients may be a novel biomarker for GBM diagnosis and that treatment targeting microRNA-1246 may contribute to antitumor immunotherapy.

TGFß1-induced beta-site APP-cleaving enzyme 2 upregulation promotes tumorigenesis through the NF-κB signalling pathway in human gliomas.

Gliomas are the most common primary malignant tumours of the central nervous system, and new molecular biomarkers are urgently needed for diagnosis and targeted therapy. Here, we report that increased beta-site APP-cleaving enzyme 2 (BACE2) expression is associated with increases in the grade of human glioma, the incidence of the mesenchymal molecular glioblastoma multiforme subtype and the likelihood of poor prognoses for patients. BACE2 knockdown suppressed cell invasion, cell migration and tumour growth both in vitro and in vivo, while BACE2 overexpression promoted the mesenchymal transition and cell proliferation. Furthermore, TGFß1 stimulated BACE2 expression through Smad-dependent signalling, which modulated TNF-α-induced NF-κB activity through the PP1A/IKK pathway to promote tumorigenesis in both U87MG and U251 cells. Our study indicated that BACE2 plays a significant role in glioma development. Therefore, BACE2 is a potential therapeutic target for human gliomas due to its function and ability to be regulated.

PLEKHG5 is a novel prognostic biomarker in glioma patients.

BACKGROUND: PLEKHG5, a Rho-specific guanine-nucleotide exchange factor, is involved in tumor cell migration, invasion and angiogenic potential. In this study, the expression pattern, prognostic value and function of PLEKHG5 in gliomas were investigated. METHODS: Immunohistochemistry was used to determine the expression pattern of PLEKHG5 in 61 glioma patients after curative resection. Statistical analysis was performed to evaluate the diagnostic and prognostic significance of PLEKHG5. Gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis and Gene set enrichment analysis (GSEA) were used to predict potential functions of PLEKHG5. Migration assay and western blot analysis determined PLEKHG5 function in glioma migration and invasion. RESULTS: Increased PLEKHG5 expression levels were associated with higher glioma grades (P < 0.05). In addition, glioblastomas multiforme have higher ratio and stronger intensity of PLEKHG5 expression compared with low-grade gliomas. High expression level of PLEKHG5 indicated poorer prognosis and shorter survival time in all glioma patients (P < 0.001). GO analysis, KEGG pathway analysis and GSEA analysis suggested that PLEKHG5 was involved in glioma migration, invasion and epithelial-mesenchymal transition. Migration assay and western blot analysis revealed PLEKHG5 promoted glioma migration and invasion. CONCLUSION: Our results demonstrated PLEKHG5 could be used as a novel prognostic biomarker and anti-tumor target for glioma patients.

Mucin O-glycosylating enzyme GALNT2 facilitates the malignant character of glioma by activating the EGFR/PI3K/Akt/mTOR axis.

N-Acetylgalactosaminyltransferase 2 (GALNT2), the enzyme that regulates the initial step of mucin O-glycosylation, has been reported to play a role in influencing the malignancy of various cancers. However, the mechanism through which it influences gliomas is still unknown. In the current study, the Cox proportional hazards model was used to select genes. Data obtained from The Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC) of clinical specimens showed that increased GALNT2 expression levels were associated with an unfavorable prognosis and a higher tumor grade in human gliomas. Then, GALNT2 knockdown and overexpression were performed in glioma cell lines and verified by quantitative real-time PCR (qRT-PCR) and Western blotting. Functional assays demonstrated that GALNT2 was closely related to glioma cell proliferation, cycle transition, migration and invasion. Western blot analysis and lectin pull-down assays indicated that GALNT2 knockdown decreased the level of phosphorylated epidermal growth factor receptor (EGFR) and the expression of the Tn antigen on EGFR and affected the expression levels of p21, cyclin-dependent kinase 4 (CDK4), cyclinD1, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) through the EGFR/PI3K/Akt/mTOR pathway. GALNT2 overexpression had the opposite effects. In vivo, the growth of orthotopic glioma xenografts in nude mice was distinctly inhibited by the expression of GALNT2 shRNA, and the tumors with GALNT2 shRNA exhibited less aggressiveness and reduced expression of Ki67 and MMP2. Overall, GALNT2 facilitates the malignant characteristics of glioma by influencing the O-glycosylation and phosphorylation of EGFR and the subsequent downstream PI3K/Akt/mTOR axis. Therefore, GALNT2 may serve as a novel biomarker and a potential target for future therapy of glioma.

Efficient acetogenesis of anaerobic co-digestion of food waste and maize straw in a HSAD reactor.

In this study, food waste and maize straw were used as feedstock, and the two-phase high-solid anaerobic digestion (TP-HSAD) technology was used to optimize the process parameters of leachate reflux in acid-production stage. Results indicated that compared with other waste activated sludge, pig manure digestate (PM) as leachate can achieve better hydrolysis and acidification effect. The increase of leachate reflux ratio can shorten the fermentation time of the acid-producing stage and increase the fermentation efficiency. When the reflux ratio was 32:1, peak concentration of volatile fatty acids (VFAs) was 45.4 g/L and the volatile solids (VS) removal rate was 61.7%. Reflux frequency has minimal effect on the concentration of VFAs and the degree of degradation of VS, but a higher reflux frequency will prolong the reaction time of acid-production stage. When PM is used as reflux leachate, the HSAD reactor can improve the hydrolysis and acidification of the anaerobic fermentation.

Exosomes derived from mesenchymal stem cells attenuate the progression of atherosclerosis in ApoE-/- mice via miR-let7 mediated infiltration and polarization of M2 macrophage.

Atherosclerosis is a chronic inflammatory disease of the vasculature. Exosomes derived from mesenchymal stem cells (MSCs) exert immunomodulatory and immunosuppressive effects; however, the MSCs-exosomes administration on atherosclerosis was unknown. Here, our ApoE-/- mice were fed a high-fat diet and received intravenous injections of exosomes from MSCs for 12 weeks. After tail-vein injection, MSCs-exosomes were capable of migrating to atherosclerotic plaque and selectively taking up residence near macrophages. MSCs-exosomes treatment decreased the atherosclerotic plaque area of ApoE-/- mice and greatly reduced the infiltration of macrophages in the plaque, associating induced macrophage polarization towards M2. In vitro, MSCs-exosomes treatment markedly inhibited LPS-induced M1 markers expression, while increased M2 markers expression in macrophages. Moreover, miR-let7 family was found to be highly enriched in MSCs-exosomes. Endogenous miR-let7 expression was found in the aortic root of ApoE-/- mice, and MSCs-exosomes treatment further up-regulated miR-let7 levels. In addition, inhibition of miR-let7 in U937 cells significantly inhibited the migration and M2 polarization via IGF2BP1 and HMGA2 pathway respectively in vitro. Our study demonstrates that MSCs-exosomes ameliorated atherosclerosis in ApoE-/- and promoted M2 macrophage polarization in the plaque through miR-let7/HMGA2/NF-κB pathway. In addition, MSCs-exosomes suppressed macrophage infiltration via miR-let7/IGF2BP1/PTEN pathway in the plaque. This finding extends our knowledge on MSCs-exosomes affect inflammation in atherosclerosis plaque and provides a potential method to prevent the atherosclerosis. Exosomes from MSCs hold promise as therapeutic agents to reduce the residual risk of coronary artery diseases.

GALE Promotes the Proliferation and Migration of Glioblastoma Cells and Is Regulated by miR-let-7i-5p.

PURPOSE: Glioma is the most common and lethal type of brain tumor. While GALE (UDP-galactose-4-epimerase) has been shown to be overexpressed in some kinds of cancers, its expression in gliomas has not been reported. MicroRNAs (miRNAs) function as tumor suppressors in many cancers, and online datasets can be used to predict whether GALE is regulated by miR-let-7i-5p. In this investigation, we explored the effect and regulatory mechanisms of GALE on glioblastoma growth via miR-let-7i-5p. METHODS: We used a Cox proportional hazards model and publicly available datasets to examine the relationship between GALE and the survival rates of glioma patients. Bioinformatics predicted the targeting of GALE by miR-let-7i-5p. The proliferation, migration, cell cycle and apoptosis of human glioblastoma cells were assessed by relevant assays. We also demonstrated the effect of GALE on glioblastoma multiforme [GBM] tumor growth using an in vivo orthotopic xenograft model. RESULTS: GALE was upregulated in human gliomas, especially in high-grade gliomas (e.g., GBM). An obvious decline in GALE expression was observed in human glioblastoma cell lines (U87 and U251) following treatment with a small interfering RNA (siRNA) targeting GALE or miR-let-7i-5p mimics. Knockdown of GALE or overexpression of miR-let-7i-5p (with miR-let-7i-5p mimics) inhibited U87 and U251 cell growth. miR-let-7i-5p significantly restrained the migration ability of human glioblastoma cells in vascular mimic (VM), wound healing and transwell assays, and GALE promoted glioblastoma growth in vivo. CONCLUSION: Our findings confirm that GALE plays an important role in promoting the development of human glioma and that GALE can be regulated by miR-let-7i-5p to inhibit human glioblastoma growth. IMPLICATIONS FOR CANCER SURVIVORS: Our data show that cancer survivors have low GALE expression, which indicates that GALE may be a diagnostic biomarker and a promising therapeutic target in glioblastoma.

Glioma exosomes mediate the expansion and function of myeloid-derived suppressor cells through microRNA-29a/Hbp1 and microRNA-92a/Prkar1a pathways.

Myeloid-derived suppressor cells (MDSCs) play a pivotal role in mediating the formation of an immunosuppressive environment and assisting tumors in evading the host immune response. However, the mechanism through which tumors manipulate the differentiation and function of MDSCs remains unclear. Here, we report that hypoxia-induced glioma cells can stimulate the differentiation of functional MDSCs by transferring exosomal miR-29a and miR-92a to MDSCs. Our results showed that glioma-derived exosomes (GEXs) can enhance the differentiation of functional MDSCs both in vitro and in vivo, and hypoxia-induced GEXs (H-GEXs) demonstrated a stronger MDSCs induction ability than did normoxia-induced GEXs (N-GEXs). A subsequent miRNA sequencing analysis of N-GEXs and H-GEXs revealed that hypoxia-induced exosomal miR-29a and miR-92a expression induced the propagation of MDSCs. miR-29a and miR-92a activated the proliferation and function of MDSCs by targeting high-mobility group box transcription factor 1 (Hbp1) and protein kinase cAMP-dependent type I regulatory subunit alpha (Prkar1a), respectively. Altogether, the results of our study provide new insights into the role of glioma exosomal miRNAs in mediating the formation of immunosuppressive microenvironments in tumors and elucidate the underlying exosomal miR-29a/miR-92a-based regulatory mechanism responsible for the modulation of functional MDSC induction.

Immunosuppressive effects of hypoxia-induced glioma exosomes through myeloid-derived suppressor cells via the miR-10a/Rora and miR-21/Pten Pathways.

While immunosuppressive environments mediated by myeloid-derived suppressor cells (MDSCs) have been well documented in glioma patients, the mechanisms of MDSC development and activation have not been clearly defined. Here, we elucidated a role for glioma-derived exosomes (GDEs) in potentiating an MDSC pathway. We isolated normoxia-stimulated and hypoxia-stimulated GDEs and studied their MDSC induction abilities in vivo and in vitro. Analyses of spleen and bone marrow MDSC proportions (flow cytometry) and reactive oxygen species (ROS), arginase activity, nitric oxide (NO), T-cell proliferation and immunosuppressive cytokine (IL-10 and TGF-ß, ELISA) levels were used to assess MDSC expansion and functional capacity. We also performed microRNA (miRNA) sequencing analysis of two types of GDEs to find miRNAs that potentially mediate the development and activation of MDSCs. GDE miRNA intracellular signaling in MDSCs was also studied. Hypoxia promoted the secretion of GDEs, and mouse MDSCs could uptake GDEs. Hypoxia-stimulated GDEs had a stronger ability to induce MDSCs than N-GDEs. The hypoxia-inducible expression of miR-10a and miR-21 in GDEs mediated GDE-induced MDSC expansion and activation by targeting RAR-related orphan receptor alpha (RORA) and phosphatase and tensin homolog (PTEN). Mice inoculated with miR-10a or miR-21 knockout glioma cells generated fewer MDSCs than those inoculated with normal glioma cells. These data elucidated a mechanism by which glioma cells influence the differentiation and activation of MDSCs via exosomes and demonstrated how local glioma hypoxia affects the entirety of tumor immune environments.