Glioma stem cell-derived exosomes induce the transformation of astrocytes via the miR-3065-5p/DLG2 signaling axis.

Tumor-associated astrocytes (TAAs) in the glioblastoma microenvironment play an important role in tumor development and malignant progression initiated by glioma stem cells (GSCs). In the current study, normal human astrocytes (NHAs) were cultured and continuously treated with GSC-derived exosomes (GSC-EXOs) induction to explore the mechanism by which GSCs affect astrocyte remodeling. This study revealed that GSC-EXOs can induce the transformation of NHAs into TAAs, with relatively swollen cell bodies and multiple extended processes. In addition, high proliferation, elevated resistance to temozolomide (TMZ), and increased expression of TAA-related markers (TGF-ß, CD44, and tenascin-C) were observed in the TAAs. Furthermore, GSC-derived exosomal miR-3065-5p could be delivered to NHAs, and miR-3065-5p levels increased significantly in TAAs, as verified by miRNA expression profile sequencing and Reverse transcription polymerase chain reaction. Overexpression of miR-3065-5p also enhanced NHA proliferation, elevated resistance to TMZ, and increased the expression levels of TAA-related markers. In addition, both GSC-EXO-induced and miR-3065-5p-overexpressing NHAs promoted tumorigenesis of GSCs in vivo. Discs Large Homolog 2 (DLG2, downregulated in glioblastoma) is a direct downstream target of miR-3065-5p in TAAs, and DLG2 overexpression could partially reverse the transformation of NHAs into TAAs. Collectively, these data demonstrate that GSC-EXOs induce the transformation of NHAs into TAAs via the miR-3065-5p/DLG2 signaling axis and that TAAs can further promote the tumorigenesis of GSCs. Thus, precisely blocking the interactions between astrocytes and GSCs via exosomes may be a novel strategy to inhibit glioblastoma development, but more in-depth mechanistic studies are still needed.

TBX15 facilitates malignant progression of glioma by transcriptional activation of TXDNC5.

T-box transcription factor 15 (TBX15) plays important role in various cancers; however, its expression and role in glioma is still unclear. In this study, our findings indicated that TBX15 was increased in gliomas compared to normal brain tissues, and high levels of TBX15 were related to poor survival. Furthermore, TBX15 silencing in glioma cells not only inhibited their proliferation, migration, and invasion in vitro, but also weakened their ability to recruit macrophages and polarize the latter to the M2 subtype. Mechanism study indicated that thioredoxin domain containing 5 (TXNDC5) lies downstream of TBX15. Furthermore, rescue assays verified that the role of TBX15 in glioma cells is dependent on TXNDC5. Moreover, sh-TBX15 loaded into DNA origami nanocarrier suppressed the malignant phenotype of glioma in vitro and in vivo. Taken together, the TBX15/TXNDC5 axis is involved in the genesis and progression of glioma, and is a potential therapeutic target.

Transformed astrocytes confer temozolomide resistance on glioblastoma via delivering ALKBH7 to enhance APNG expression after educating by glioblastoma stem cells-derived exosomes.

BACKGROUND: Glioblastoma is the most malignant primary brain tumor in adults. Temozolomide (TMZ) stands for the first-line chemotherapeutic agent against glioblastoma. Nevertheless, the therapeutic efficacy of TMZ appears to be remarkably limited, because of low cytotoxic efficiency against glioblastoma. Besides, various mechanical studies and the corresponding strategies fail to enhancing TMZ curative effect in clinical practice. Our previous studies have disclosed remodeling of glial cells by GSCs, but the roles of these transformed cells on promoting TMZ resistance have never been explored. METHODS: Exosomes were extracted from GSCs culture through standard centrifugation procedures, which can activate transformation of normal human astrocytes (NHAs) totumor-associated astrocytes (TAAs) for 3 days through detect the level of TGF-ß, CD44 and tenascin-C. The secretive protein level of ALKBH7 of TAAs was determined by ELISA kit. The protein level of APNG and ALKBH7 of GBM cells were determined by Western blot. Cell-based assays of ALKBH7 and APNG triggered drug resistance were performed through flow cytometric assay, Western blotting and colony formation assay respectively. A xenograft tumor model was applied to investigate the function of ALKBH7 in vivo. Finally, the effect of the ALKBH7/APNG signaling on TMZ resistance were evaluated by functional experiments. RESULTS: Exosomes derived from GSCs can activate transformation of normal human astrocytes (NHAs)to tumor-associated astrocytes (TAAs), as well as up-regulation of ALKBH7expression in TAAs. Besides, TAAs derived ALKBH7 can regulate APNG gene expression of GBM cells. After co-culturing with TAAs for 5 days, ALKBH7 and APNG expression in GBM cells were elevated. Furthermore, Knocking-down of APNG increased the inhibitory effect of TMZ on GBM cells survival. CONCLUSION: The present study illustrated a new mechanism of glioblastoma resistance to TMZ, which based on GSCs-exo educated TAAs delivering ALKBH7 to enhance APNG expression of GBM cells, which implied that targeting on ALKBH7/APNG regulation network may provide a new strategy of enhancing TMZ therapeutic effects against glioblastoma.

Hsa_circ_0021205 enhances lipolysis via regulating miR-195-5p/HSL axis and drives malignant progression of glioblastoma.

Abnormal lipid metabolism is an essential hallmark of glioblastoma. Hormone sensitive lipase (HSL), an important rate-limiting enzyme contributed to lipolysis, which was involved in aberrant lipolysis of glioblastoma, however, its definite roles and the relevant regulatory pathway have not been fully elucidated. Our investigations disclosed high expression of HSL in glioblastoma. Knock-down of HSL restrained proliferation, migration, and invasion of glioblastoma cells while adding to FAs could significantly rescue the inhibitory effect of si-HSL on tumor cells. Overexpression of HSL further promoted tumor cell proliferation and invasion. Bioinformatics analysis and dual-luciferase reporter assay were performed to predict and verify the regulatory role of ncRNAs on HSL. Mechanistically, hsa_circ_0021205 regulated HSL expression by sponging miR-195-5p, which further promoted lipolysis and drove the malignant progression of glioblastoma. Besides, hsa_circ_0021205/miR-195-5p/HSL axis activated the epithelial-mesenchymal transition (EMT) signaling pathway. These findings suggested that hsa_circ_0021205 promoted tumorigenesis of glioblastoma through regulation of HSL, and targeting hsa_circ_0021205/miR-195-5p/HSL axis can serve as a promising new strategy against glioblastoma.

Glioblastoma stem cell-derived exosomal miR-374b-3p promotes tumor angiogenesis and progression through inducing M2 macrophages polarization.

Glioblastoma stem cells (GSCs) reside in hypoxic periarteriolar niches of glioblastoma micro-environment, however, the crosstalk of GSCs with macrophages on regulating tumor angiogenesis and progression are not fully elucidated. GSCs-derived exosomes (GSCs-exos) are essential mediators during tumor immune-microenvironment remodeling initiated by GSCs, resulting in M2 polarization of tumor-associated macrophages (TAMs) as we reported previously. Our data disclosed aberrant upregulation of miR-374b-3p in both clinical glioblastoma specimens and human cell lines of GSCs. MiR-374b-3p level was high in GSCs-exos and can be internalized by macrophages. Mechanistically, GSCs exosomal miR-374b-3p induced M2 polarization of macrophages by downregulating phosphatase and tensin expression, thereby promoting migration and tube formation of vascular endothelial cells after coculture with M2 macrophages. Cumulatively, these data indicated that GSCs exosomal miR-374b-3p can enhance tumor angiogenesis by inducing M2 polarization of macrophages, as well as promote malignant progression of glioblastoma. Targeting exosomal miR-374b-3p may serve as a potential target against glioblastoma.

HDAC1 mediates epithelial-mesenchymal transition and promotes cancer cell invasion in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most malignant tumors of the central nervous system, and its treatment has always been a difficult clinical problem. Here, we evaluated HDAC1 expression patterns and their effect on prognosis based on GBM cases from TCGA and CGGA databases. Expression was compared between GBM samples and normal controls. High HDAC1 expression was found to be an indicator of poor prognosis in glioblastoma. We also established a protein-protein interaction network to explore HDAC1-related interacting proteins, including the epithelial-mesenchymal transition (EMT)-related protein VIM, which is closely associated with HDAC1. Consistently, functional enrichment analysis showed that several GBM tissues with high HDAC1 were enriched in the expression of cancer markers, such as those involved in glycolysis, hypoxia, inflammation, and some signaling pathways. Next, this study analyzed the effect of HDAC1 on invasive ability and the EMT signaling pathway in GBM cells in vitro. The results showed that an HDAC1 inhibitor (RGFP109) could inhibit the EMT process in glioma cells in vitro, thereby affecting the invasion and migration of cells. Similar results were obtained based on in vivo studies. Our data suggest that HDAC1 has the potential to be a powerful prognostic biomarker, which might provide a basis for developing therapeutic targets for GBM.

Downregulated M6A modification and expression of circRNA_103239 promoted the progression of glioma by regulating the miR-182-5p/MTSS1 signalling pathway.

Glioma is a common type of tumor in the central nervous system, and the mortality is high. The prognosis of advanced glioma patients remains poor, and the therapeutic strategies need to be developed. The roles of circRNAs in glioma remain largely unknown. The aim of this study was to explore the functions circRNA_103239 in the biological behaviour changes of glioma cells. The expression of circRNA_103239 in clinical samples and glioma cells were examined using RT-qPCR. The targets of circRNA_103239 were predicted using bioinformatics approach. Gain- and loss-of-function study were carried out. The proliferation of transfected cells were evaluated by CCK-8 assay. Migratory and invasive activities of the cells were examined using wound healing, colony formation and transwell assay. Tumor growth was also evaluated in vivo. The results indicated that the expression of circRNA_103239 was predominantly detected in the cytoplasma of glioma cells. In addition, the expression of circRNA_103239 was down-regulated in glioma, and up-regulated circRNA_103239 inhibited the progression of glioma. Furthermore, miR-182-5p was the novel target of circRNA_103239 in glioma, and MTSS1 was the putative downstream molecule of circRNA_103239/miR-182-5p axis. Additionally, circRNA_103239 suppressed the progression of glioma in a miR-182-5p/MTSS1 dependent manner. Moreover, circRNA_103239 inhibited tumour growth in vivo, and the expression of circRNA_103239 was regulated by METTL14-mediated m6A modification. In summary, in normal cells, METTL14 mediated the m6A modification and expression of circRNA_103239, which sponging miR-182-5p and inducing the expression of MTSS1, subsequently inhibiting the EMT; whereas in glioma cells, downregulated METTL14 induced downregulated m6A modification and expression of circRNA_103239, further resulting in the up-regulation of miR-182-5p and down-regulation of MTSS1, consequently promoting the EMT of glioma cells and triggering the progression of tumor.

Long noncoding RNA HOXC-AS3 remodels lipid metabolism and promotes the proliferation of transformed macrophages in the glioma stem cell microenvironment by regulating the hnRNPA1/CaM axis.

Metabolism remodelling of macrophages in the glioblastoma microenvironment contributes to immunotherapeutic resistance. However, glioma stem cell (GSC)-initiated lipid metabolism remodelling of transformed macrophages (tMΦs) and its effect on the glioblastoma microenvironment have not been fully elucidated. Total cholesterol (TC) levels and lipid metabolism enzyme expression in macrophages in the GSC microenvironment were evaluated and found that the TC levels of tMΦs were increased, and the expression of the lipid metabolism enzymes calmodulin (CaM), apolipoprotein E (ApoE), and liver X receptor (LXR) was upregulated. Knockdown of HOXC-AS3 led to a decrease in the proliferation, colony formation, invasiveness, and tumorigenicity of tMΦs. Downregulation of CaM resulted in a decline in TC levels. HOXC-AS3 overexpression led to increases in both CaM expression levels and TC levels in tMΦs. RNA pull down and mass spectrometry experiments were conducted and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was screened as the HOXC-AS3 binding proteins related to lipid metabolism. RIP and RNA pull down assays verified that HOXC-AS3 can form a complex with hnRNPA1. Knockdown of hnRNPA1 downregulated CaM expression; however, downregulation of HOXC-AS3 did not affect hnRNPA1 expression.TMΦs underwent lipid metabolism remodelling induced by GSC via the HOXC-AS3/hnRNPA1/CaM pathway, which enhanced the protumor activities of tMΦs, and may serve as a potential metabolic intervening target to improve glioblastoma immunotherapy.

Upregulation of the ZNF148/PTX3 axis promotes malignant transformation of dendritic cells in glioma stem-like cells microenvironment.

BACKGROUND: The recent development of dendritic cell (DC)-based immunotherapy has resulted in advances in glioblastoma multiforme (GBM) treatment. However, the cell fate of DCs in the GBM microenvironment, especially in microenvironments in which glioma stem cell (GSCs)-mediated remodeling has resulted in highly immunosuppressive conditions, has not yet been fully investigated. METHODS: Observed the interaction between GSCs and primary cultured DCs in a dual-color tracing model, monoclonal and continuously passaged highly proliferative DCs, and named transformed DCs (t-DCs). The expression of DC-specific surface markers was analyzed using RT-PCR, chromosome karyotype, and flow cytometry. The expression of long pentraxin 3 (PTX3) and its transcription factor zinc finger protein 148 (ZNF148) in t-DCs was detected using qRT-PCR and western blot. CCK8 and transwell assays were conducted to assess the effect of ZNF148 and PTX3 on the proliferation, migration, and invasion of t-DCs. Bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP)-qPCR assay were used to explore the relation between ZNF148 and PTX3. RESULTS: Transformed DCs (t-DCs) still expressed DC-specific surface markers, namely, CD80 and CD11c, and immune-related costimulatory molecules, namely, CD80, CD86, CD40, and ICAM-1. However, the expression levels of these molecules in t-DCs decreased moderately compared to those in naive DCs. Stable overexpression of PTX3 further promoted the proliferation and migration of t-DCs in vitro, decreased the expression of costimulatory molecules, and increased the tumorigenicity of t-DCs in vivo. The transcription factor zinc finger protein 148 (ZNF148) was directly bound to the PTX3 promoter region and enhanced PTX3 expression. Downregulation of ZNF148 significantly decreased PTX3 expression and reduced the proliferation and migration of t-DCs. Overexpression of ZNF148 significantly increased PTX3 expression and promoted the proliferation and migration of t-DCs, achieving the same biological effects as PTX3 overexpression in t-DCs. Simultaneously, the downregulation of ZNF148 partially reversed the effect of PTX3 overexpression in t-DCs. CONCLUSION: The ZNF148/PTX3 axis played an important role in regulating the malignant transformation of DCs after cross-talk with GSCs, and this axis may serve as a new target for sensitizing GBM to DC-based immunotherapy.

RNA-binding protein DHX9 promotes glioma growth and tumor-associated macrophages infiltration via TCF12.

BACKGROUND: Glioma is the most common malignant tumor of the central nervous system, with high heterogeneity, strong invasiveness, high therapeutic resistance, and poor prognosis, comprehending a serious challenge in neuro-oncology. Until now, the mechanisms underlying glioma progression have not been fully elucidated. METHODS: The expression of DExH-box helicase 9 (DHX9) in tissues and cells was detected by qRT-PCR and western blot. EdU and transwell assays were conducted to assess the effect of DHX9 on proliferation, migration and invasion of glioma cells. Cocultured model was used to evaluate the role of DHX9 on macrophages recruitment and polarization. Animal study was performed to explore the role of DHX9 on macrophages recruitment and polarization in vivo. Bioinformatics analysis, dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay was used to explore the relation between DHX9 and TCF12/CSF1. RESULTS: DHX9 was elevated in gliomas, especially in glioblastoma multiforme (GBM). Besides promoting the proliferation, migration, and invasion of glioma cells, DHX9 facilitated the infiltration of macrophages into glioma tissues and polarization to M2-like macrophages, known as tumor-associated macrophages (TAMs). DHX9 silencing decreased the expression of colony-stimulating factor 1 (CSF1), which partially restored the inhibitory effect on malignant progress of glioma and infiltration of TAMs caused by DHX9 knockdown by targeting the transcription factor 12 (TCF12). Moreover, TCF12 could directly bind to the promoter region of CSF1. CONCLUSION: DHX9/TCF12/CSF1 axis regulated the increases in the infiltration of TAMs to promote glioma progression and might be a novel potential target for future immune therapies against gliomas.

Exosomal miR-6733-5p mediates cross-talk between glioblastoma stem cells and macrophages and promotes glioblastoma multiform progression synergistically.

AIM: Exosomal miRNAs derived from glioblastoma stem cells (GSCs) are important mediators of immunosuppressive microenvironment formation in glioblastoma multiform (GBM), especially in M2-like polarization of tumor-associated macrophages (TAMs). However, the exact mechanisms by which GSCs-derived exosomes (GSCs-exo) facilitate the remodeling of the immunosuppressive microenvironment of GBM have not been elucidated. METHODS: Transmission electron microscopy (TME) and nanoparticle tracking analysis (NTA) were applied to verify the existence of GSCs-derived exosomes. Sphere formation assays, flow cytometry, and tumor xenograft transplantation assays were performed to identify the exact roles of exosomal miR-6733-5p. Then, the mechanisms of miR-6733-5p and its downstream target gene regulating crosstalk between GSCs cells and M2 macrophages were further investigated. RESULTS: GSCs-derived exosomal miR-6733-5p induce macrophage M2 polarization of TAMs by positively targeting IGF2BP3 to activate the AKT signaling pathway, which further facilitates the self-renewal and stemness of GSCs. CONCLUSION: GSCs secrete miR-6733-5p-rich exosomes to induce M2-like polarization of macrophages, as well as enhance GSCs stemness and promote malignant behaviors of GBM through IGF2BP3 activated AKT pathway. Targeting GSCs exosomal miR-6733-5p may provide a potential new strategy against GBM.