m5C modification of LINC00324 promotes angiogenesis in glioma through CBX3/VEGFR2 pathway.

Angiogenesis plays a major role in tumor initiation, progression, and metastasis. This is why finding antiangiogenic targets is essential in the treatment of gliomas. In this study, NSUN2 and LINC00324 were significantly upregulated in conditionally cultured glioblastoma endothelial cells (GECs). Knockdown of NSUN2 or LINC00324 inhibits GECs angiogenesis. NSUN2 increased the stability of LINC00324 by m5C modification and upregulated LINC00324 expression. LINC00324 competes with the 3'UTR of CBX3 mRNA to bind to AUH protein, reducing the degradation of CBX3 mRNA. In addition, CBX3 directly binds to the promoter region of VEGFR2, enhances VEGFR2 transcription, and promotes GECs angiogenesis. These findings demonstrated NSUN2/LINC00324/CBX3 axis plays a crucial role in regulating glioma angiogenesis, which provides new strategies for glioma therapy.

RBMS3-induced circHECTD1 encoded a novel protein to suppress the vasculogenic mimicry formation in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly vascularized malignant cancer of the central nervous system, and the presence of vasculogenic mimicry (VM) severely limits the effectiveness of anti-vascular therapy. In this study, we identified downregulated circHECTD1, which acted as a key VM-suppressed factor in GBM. circHECTD1 elevation significantly inhibited cell proliferation, migration, invasion and tube-like structure formation in GBM. RIP assay was used to demonstrate that the flanking intron sequence of circHECTD1 can be specifically bound by RBMS3, thereby inducing circHECTD1 formation to regulate VM formation in GBM. circHECTD1 was confirmed to possess a strong protein-encoding capacity and the encoded functional peptide 463aa was identified by LC-MS/MS. Both circHECTD1 and 463aa significantly inhibited GBM VM formation in vivo and in vitro. Analysis of the 463aa protein sequence revealed that it contained a ubiquitination-related domain and promoted NR2F1 degradation by regulating the ubiquitination of the NR2F1 at K396. ChIP assay verified that NR2F1 could directly bind to the promoter region of MMP2, MMP9 and VE-cadherin, transcriptionally promoting the expression of VM-related proteins, which in turn enhanced VM formation in GBM. In summary, we clarified a novel pathway for RBMS3-induced circHECTD1 encoding functional peptide 463aa to mediate the ubiquitination of NR2F1, which inhibited VM formation in GBM. This study aimed to reveal new mechanisms of GBM progression in order to provide novel approaches and strategies for the anti-vascular therapy of GBM. The schematic illustration showed the inhibitory effect of circHECTD1-463aa in the VM formation in GBM.

SUMOylation of RALY promotes vasculogenic mimicry in glioma cells via the FOXD1/DKK1 pathway.

Human malignant gliomas are the most common and aggressive primary malignant tumors of the human central nervous system. Vasculogenic mimicry (VM), which refers to the formation of a tumor blood supply system independently of endothelial cells, contributes to the malignant progression of glioma. Therefore, VM is considered a potential target for glioma therapy. Accumulated evidence indicates that alterations in SUMOylation, a reversible post-translational modification, are involved in tumorigenesis and progression. In the present study, we found that UBA2 and RALY were upregulated in glioma tissues and cell lines. Downregulation of UBA2 and RALY inhibited the migration, invasion, and VM of glioma cells. RALY can be SUMOylated by conjugation with SUMO1, which is facilitated by the overexpression of UBA2. The SUMOylation of RALY increases its stability, which in turn increases its expression as well as its promoting effect on FOXD1 mRNA. The overexpression of FOXD1 promotes DKK1 transcription by activating its promoter, thereby promoting glioma cell migration, invasion, and VM. Remarkably, the combined knockdown of UBA2, RALY, and FOXD1 resulted in the smallest tumor volumes and the longest survivals of nude mice in vivo. UBA2/RALY/FOXD1/DKK1 axis may play crucial roles in regulating VM in glioma, which may contribute to the development of potential strategies for the treatment of gliomas.

Pseudogene MAPK6P4-encoded functional peptide promotes glioblastoma vasculogenic mimicry development.

Glioma is the most common primary malignancy of the central nervous system. Glioblastoma (GBM) has the highest degree of malignancy among the gliomas and the strongest resistance to chemotherapy and radiotherapy. Vasculogenic mimicry (VM) provides tumor cells with a blood supply independent of endothelial cells and greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. Vascular endothelial growth factor receptor 2 (VEGFR2) and vascular endothelial cadherin (VE-cadherin) are currently recognized molecular markers of VM in tumors. In the present study, we show that pseudogene MAPK6P4 deficiency represses VEGFR2 and VE-cadherin protein expression levels, as well as inhibits the proliferation, migration, invasion, and VM development of GBM cells. The MAPK6P4-encoded functional peptide P4-135aa phosphorylates KLF15 at the S238 site, promoting KLF15 protein stability and nuclear entry to promote GBM VM formation. KLF15 was further confirmed as a transcriptional activator of LDHA, where LDHA binds and promotes VEGFR2 and VE-cadherin lactylation, thereby increasing their protein expression. Finally, we used orthotopic and subcutaneous xenografted nude mouse models of GBM to verify the inhibitory effect of the above factors on GBM VM development. In summary, this study may represent new targets for the comprehensive treatment of glioma.

CircMTA1 promotes glioblastoma angiogenesis by encoding MTA1-134aa.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with rapid angiogenesis. How to inhibit GBM angiogenesis is a key problem to be solved. To explore the targets of inhibiting GBM angiogenesis, this study confirmed that the expression of circMTA1 (hsa_circ_0033614) was significantly upregulated in human brain microvascular endothelial cells exposed to glioma cell-conditioned medium (GECs). The expression of circMTA1 in the cytoplasm was significantly higher than that in the nucleus. Upregulated circMTA1 in GECs can promote cell proliferation, migration, and tube formation. Further exploration of the circularization mechanism of circMTA1 confirmed that KHDRBS1 protein can bind to the upstream and downstream flanking sequences of circMTA1 and promote circMTA1 biogenesis by coordinating Alu element pairing. KHDRBS1 upregulated the proliferation, migration, and tube formation of GECs by promoting the biogenesis of circMTA1. CircMTA1 can encode the protein MTA1-134aa by internal ribosome entry site sequence-mediated translation mechanism, and promote the proliferation, migration, and tube formation of GECs through the encoded MTA1-134aa. This study provides a new target for inhibiting angiogenesis in brain GBM and a new strategy for improving the therapeutic efficacy of GBM.

A novel peptide P1-121aa encoded by STK24P1 regulates vasculogenic mimicry via ELF2 phosphorylation in glioblastoma.

Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Vasculogenic mimicry (VM) is a hematological system composed of tumor cells that exert blood perfusion without relying on vascular endothelial cells. The current poor results of anti-vascular therapy for clinical GBM are associated with the presence of VM; therefore, it is important to investigate VM formation in GBM. Our results demonstrate that STK24P1 encodes P1-121aa with a kinase structural domain, and in vitro kinase assays demonstrated that P1-121aa mediates modification of ELF2 phosphorylation. ChIP and dual luciferase reporter gene assays demonstrated that the transcription factor ELF2 binds to VE-cadherin and the VEGFR2 promoter region, thereby promoting VM formation in glioma cells. P1-121aa, encoded by the pseudogene STK24P1, phosphorylates ELF2 at S107, increasing the stability of the ELF2 protein. ELF2 promotes VEGFR2 and VE-cadherin expression at the transcriptional level, which in turn promotes VM in GBM. This study demonstrates the important roles of STK24P1, P1-121aa, and ELF2 in regulating VM in GBM, which could provide potential targets for GBM treatment.

U3 snoRNA-mediated degradation of ZBTB7A regulates aerobic glycolysis in isocitrate dehydrogenase 1 wild-type glioblastoma cells.

AIMS: The isocitrate dehydrogenase (IDH) phenotype is associated with reprogrammed energy metabolism in glioblastoma (GBM) cells. Small nucleolar RNAs (snoRNAs) are known to exert an important regulatory role in the energy metabolism of tumor cells. The purpose of this study was to investigate the role of C/D box snoRNA U3 and transcription factor zinc finger and BTB domain-containing 7A (ZBTB7A) in the regulation of aerobic glycolysis and the proliferative capacity of IDH1 wild-type (IDH1WT ) GBM cells. METHODS: Quantitative reverse transcription PCR and western blot assays were utilized to detect snoRNA U3 and ZBTB7A expression. U3 promoter methylation status was analyzed via bisulfite sequencing and methylation-specific PCR. Seahorse XF glycolysis stress assays, lactate production and glucose consumption measurement assays, and cell viability assays were utilized to detect glycolysis and proliferation of IDH1WT GBM cells. RESULTS: We found that hypomethylation of the CpG island in the promoter region of U3 led to the upregulation of U3 expression in IDH1WT GBM cells, and the knockdown of U3 suppressed aerobic glycolysis and the proliferation ability of IDH1WT GBM cells. We found that small nucleolar-derived RNA (sdRNA) U3-miR, a small fragment produced by U3, was able to bind to the ZBTB4 3'UTR region and reduce ZBTB7A mRNA stability, thereby downregulating ZBTB7A protein expression. Furthermore, ZBTB7A transcriptionally inhibited the expression of hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA), which are key enzymes of aerobic glycolysis, by directly binding to the HK2 and LDHA promoter regions, thereby forming the U3/ZBTB7A/HK2 LDHA pathway that regulates aerobic glycolysis and proliferation of IDH1WT GBM cells. CONCLUSION: U3 enhances aerobic glycolysis and proliferation in IDH1WT GBM cells via the U3/ZBTB7A/HK2 LDHA axis.

SNORD17-mediated KAT6B mRNA 2'-O-methylation regulates vasculogenic mimicry in glioblastoma cells.

Glioblastoma (GBM) is a primary tumor in the intracranial compartment. Vasculogenic mimicry (VM) is a process in which a pipeline of tumor cells that provide blood support to carcinogenic cells is formed, and studying VM could provide a new strategy for clinical targeted treatment of GBM. In the present study, we found that SNORD17 and ZNF384 were significantly upregulated and promoted VM in GBM, whereas KAT6B was downregulated and inhibited VM in GBM. RTL-P assays were performed to verify the 2'-O-methylation of KAT6B by SNORD17; IP assays were used to detect the acetylation of ZNF384 by KAT6B. In addition, the binding of ZNF384 to the promoter regions of VEGFR2 and VE-cadherin promoted transcription, as validated by chromatin immunoprecipitation and luciferase reporter assays. And finally, knockdown of SNORD17 and ZNF384 combined with KAT6B overexpression effectively reduced the xenograft tumor size, prolonged the survival time of nude mice and reduced the number of VM channels. This study reveals a novel mechanism of the SNORD17/KAT6B/ZNF384 axis in modulating VM development in GBM that may provide a new goal for the comprehensive treatment of GBM.

A novel cuproptosis-related gene signature to predict prognosis in Glioma.

Glioma is primary brain tumour with a poor prognosis. Metabolic reprogramming is a hallmark of glioma, and is critical in the development of antiglioma agents and glioma therapy. Cuproptosis is a novel form of cell death mediated by protein lipidation and highly associated with mitochondrial metabolism. However, the clinical impact of cuproptosis-related genes (CRGs) in glioma remains largely unknown. The purpose of this study is to create a new CRGs signature that can be used to predict survival and immunotherapy in glioma patients. LASSO regression analysis was applied to establish prognostic gene signatures. Furthermore, a CRGs signature-based nomogram was developed and demonstrated good predictive potential. We also analyzed the relationship of CRGs and immune infiltration and the correlation with the pathological grade of glioma. Finally, we explored the miRNA that may regulate cuproptosis-related gene FDX1. We found that miR-606 was markedly downregulated in GBM, overexpression of miR-606 can significantly inhibit aerobic glycolysis and proliferation of GBM cells. FDX1 was upregulated in GBM, knockdown of FDX1 significantly inhibit aerobic glycolysis and proliferation of GBM cells. And luciferase assay was used to verified that miR-606 binds to and regulates FDX1 mRNA. These results provide a basis for further exploring the biological mechanisms of cuproptosis. This study may provide new potential therapeutic perspectives for patients with glioma.

The mechanism of BUD13 m6A methylation mediated MBNL1-phosphorylation by CDK12 regulating the vasculogenic mimicry in glioblastoma cells.

Vasculogenic mimicry (VM) is an endothelium-independent tumor microcirculation that provides adequate blood supply for tumor growth. The presence of VM greatly hinders the treatment of glioblastoma (GBM) with anti-angiogenic drugs. Therefore, targeting VM formation may be a feasible therapeutic strategy for GBM. The research aimed to evaluate the roles of BUD13, CDK12, MBNL1 in regulating VM formation of GBM. BUD13 and CDK12 were upregulated and MBNL1 was downregulated in GBM tissues and cells. Knockdown of BUD13, CDK12, or overexpression of MBNL1 inhibited GBM VM formation. METTL3 enhanced the stability of BUD13 mRNA and upregulated its expression through m6A methylation. BUD13 enhanced the stability of CDK12 mRNA and upregulated its expression. CDK12 phosphorylated MBNL1, thereby regulating VM formation of GBM. The simultaneous knockdown of BUD13, CDK12, and overexpression of MBNL1 reduced the volume of subcutaneously transplanted tumors in nude mice and prolonged the survival period. Thus, the BUD13/CDK12/MBNL1 axis plays a crucial role in regulating VM formation of GBM and provides a potential target for GBM therapy.

CPEB2 m6A methylation regulates blood-tumor barrier permeability by regulating splicing factor SRSF5 stability.

The blood-tumor barrier (BTB) contributes to poor therapeutic efficacy by limiting drug uptake; therefore, elevating BTB permeability is essential for glioma treatment. Here, we prepared astrocyte microvascular endothelial cells (ECs) and glioma microvascular ECs (GECs) as in vitro blood-brain barrier (BBB) and BTB models. Upregulation of METTL3 and IGF2BP3 in GECs increased the stability of CPEB2 mRNA through its m6A methylation. CPEB2 bound to and increased SRSF5 mRNA stability, which promoted the ETS1 exon inclusion. P51-ETS1 promoted the expression of ZO-1, occludin, and claudin-5 transcriptionally, thus regulating BTB permeability. Subsequent in vivo knockdown of these molecules in glioblastoma xenograft mice elevated BTB permeability, promoted doxorubicin penetration, and improved glioma-specific chemotherapeutic effects. These results provide a theoretical and experimental basis for epigenetic regulation of the BTB, as well as insight into comprehensive glioma treatment.

The MBNL1/circNTRK2/PAX5 pathway regulates aerobic glycolysis in glioblastoma cells by encoding a novel protein NTRK2-243aa.

Glioblastoma multiforme (GBM) is the most common tumor of the human central nervous system. Aerobic glycolysis has been strongly related to tumor development and malignant behavior. In this study, we found that MBNL1, circNTRK2, and NTRK2-243aa were markedly downregulated and inhibited glycolysis in GBM, whereas PAX5 was upregulated and promoted glycolysis. Functionally, MBNL1 promoted the expression of circNTRK2 by binding to NTRK2 pre-mRNA, as validated using RNA pull-down and nascent RNA immunoprecipitation assays. Mass spectrometry, western blotting, and immunofluorescence staining methods were used to detect the expression of NTRK2-243aa. NTRK2-243aa-encoded by circNTRK2-phosphorylated PAX5 at Y102, leading to the attenuation of the half-life of PAX5, as validated by in vitro kinase and MG132 rescue assays. Besides, PAX5 transcriptionally facilitated the expression of PKM2 and HK2 by binding to their promoter regions, as verified by luciferase reporter and chromatin immunoprecipitation assays. Finally, overexpression of MBNL1 and circNTRK2 combined with PAX5 knockdown effectively inhibited the formation of GBM xenograft tumors and significantly prolonged the survival of orthotopic nude mice. We have delineated that the MBNL1/circNTRK2/PAX5 pathway plays a crucial role in regulating GBM glycolysis and could provide potential targets and alternative strategies for the treatment of GBM.

Retraction Notice to: Knockdown of USF1 Inhibits the Vasculogenic Mimicry of Glioma Cells via Stimulating SNHG16/miR-212-3p and linc00667/miR-429 Axis.

[This retracts the article DOI: 10.1016/j.omtn.2018.12.017.].

Erratum: PIWIL3/OIP5-AS1/miR-367-3p/CEBPA feedback loop regulates the biological behavior of glioma cells: Erratum.

[This corrects the article DOI: 10.7150/thno.21740.].

[Corrigendum] SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR­153.

Following the publication of this article, the authors have realized that they had inadvertently used the same western blotting data to show the GAPDH control western blots in Figs. 1C and 4D. In examining their original data, the authors realized that the data for Fig. 1C had been placed incorrectly in the figure. The corrected version of Fig. 1, showing the correct GAPDH bands, is shown below. The authors sincerely apologize for the error made during the preparation of this Figure, thank the Editor for granting them the opportunity to publish this Corrigendum, and regret any inconvenience that this mistake may have caused. [the original article was published in Oncology Reports 38: 3265­3277, 2017; DOI: 10.3892/or.2017.5985].

Pseudogene RPL32P3 regulates the blood-tumor barrier permeability via the YBX2/HNF4G axis.

The existence of the blood-tumor barrier (BTB) severely hinders the transport of anti-tumor drugs to brain tumor tissues. Selectively opening BTB is of great significance to improve the chemotherapy effect of glioma. Pseudogenes have been recognized as important regulators in various biologic processes. In this study, we identified that ribosomal protein L32 pseudogene 3 (RPL32P3) was highly expressed in glioma-exposed endothelial cells (GECs). Knockdown of RPL32P3 decreased the expression of tight junction-related proteins (TJPs) and increased BTB permeability. Subsequent analysis of the underlying mechanism indicated that RPL32P3 recruited lysine methyltransferase 2 A (KMT2A) to the Y-box binding protein 2 (YBX2) promoter region and mediated H3K4me3 to promote YBX2 transcription. Highly expressed YBX2 bound and stabilized hepatocyte nuclear factor 4 gamma (HNF4G) mRNA. Highly expressed HNF4G directly bound to the promoters of TJPs ZO-1, occludin and claudin-5 to promote their transcriptional activities and regulated BTB permeability. The simultaneous knockdown of RPL32P3, YBX2, and HNF4G combined with doxorubicin (DOX) increased the apoptosis of glioma cells. In conclusion, the current study indicated that RPL32P3 knockdown increased BTB permeability through the YBX2/HNF4G pathway. These findings may provide new targets for the comprehensive treatment of glioma.