Long noncoding RNA TPT1-AS1 downregulates the microRNA-770-5p expression to inhibit glioma cell autophagy and promote proliferation through STMN1 upregulation.

Through the microarray analysis, long noncoding RNA TPT1-AS1 (TPT1-AS1) was identified in the development of glioma. However, the specific effect of TPT1-AS1 on glioma autophagy in the recent years has not fully been investigated. Therefore, the purpose of our present study is to investigate the function of TPT1-AS1 on affecting autophagy of glioma cells through regulation of microRNA-770-5p (miR-770-5p)-mediated stathmin 1 (STMN1). Initially, the expression of TPT1-AS1, miR-770-5p, and STMN1 were determined in glioma cell lines, followed by the prediction and validation of their interaction. After that, the effects of TPT1-AS1, miR-770-5p, and STMN1 on the in vitro glioma cell proliferation and autophagy were assessed using EdU assay and macrophage-derived chemokine (MDC) and on the in vivo tumor development and autophagy were evaluated using a nude mouse xenograft tumor assay and immunofluorescence assay. In comparison with the normal cells, the glioma cells displayed upregulated expression of TPT1-AS1 and STMN1, but a downregulated miR-770-5p expression. miR-770-5p, which directly targeted STMN1, could be downregulated by TPT1-AS1. Subsequently, in glioma cells, TPT1-AS1 can function to competitively bind to miR-770-5p, thus regulatEing STMN1 expression. Moreover, glioma cell proliferation and autophagy could be mediated through the TPT1-AS1/miR-770-5p/STMN1 axis. From our data we conclude an inhibitory function of TPT1-AS1 in glioma cell autophagy by downregulating miR-770-5p and upregulating STMN1, which may be instrumental for the therapeutic targeting and clinical management of glioma.

Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM.

Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM.

siRNA targeting stathmin inhibits invasion and enhances chemotherapy sensitivity of stem cells derived from glioma cell lines.

Glioma is one of the most highly angiogenic tumors, and glioma stem cells (GSCs) are responsible for resistance to chemotherapy and radiotherapy, as well as recurrence after operation. Stathmin is substantial for mitosis and plays an important role in proliferation and migration of glioma-derived endothelial cells. However, the relationship between stathmin and GSCs is incompletely understood. Here we isolated GSCs from glioma cell lines U87MG and U251, and then used siRNA targeting stathmin for silencing. We showed that silencing of stathmin suppressed the proliferation, increased the apoptosis rate, and arrested the cell cycle at G2/M phase in GSCs. Silencing of stathmin in GSCs also resulted in inhibited the migration/invasion as well as the capability of vasculogenic mimicry. The susceptibility of GSCs to temozolomide was also enhanced by stathmin silencing. Our findings suggest stathmin as a potential target in GSCs for glioma treatment.

Research progress on ferroptosis in gliomas (Review).

Glioma is the most prevalent type of brain tumor characterized by a poor 5-year survival rate and a high mortality rate. Malignant gliomas are commonly treated by surgery, chemotherapy and radiotherapy. However, due to toxicity and resistance to chemoradiotherapy, these treatments can be ineffective. Anxiety and depression are highly prevalent in patients with glioma, adversely affecting disease prognosis and posing societal concerns. Ferroptosis is a type of non-apoptotic, iron-dependent cell death characterized by the accumulation of lethal reactive oxygen species produced by iron metabolism, and it serves a key role in numerous diseases. Regulation of iron phagocytosis may serve as a therapeutic strategy for the development of novel glioma treatments. The present review discusses the mechanisms underlying the occurrence and regulation of ferroptosis, its role in the genesis and evolution of gliomas, and its association with glioma-related anxiety and depression. By exploring potential targets for glioma treatment, the present review provides a theoretical basis for the development of novel therapeutic strategies against glioma.

Microvascular decompression for intermediate nerve neuralgia: a case report and literature review.

Intermediate nerve neuralgia (INN) is a rare craniofacial pain syndrome. The diagnosis of INN is challenging because of the complex ear sensory innervation that results in a clinical overlap with both trigeminal neuralgia (TN) and glossopharyngeal neuralgia (GPN). A 76-year-old woman with a remarkable medical history presented with right otalgia and mandibular pain for 7 years. Neurological examination revealed a diminished sensation in the distribution of the intermediate nerve (IN). Magnetic resonance imaging demonstrated an impression of the anterior inferior cerebellar artery (AICA) on the facial-vestibulocochlear nerve complex (VII/VIII complex). The patient underwent microvascular decompression (MVD) after long-term oral medication. We confirmed that the responsible vessel was close to the VII/VIII complex and isolated the vessel under the microscope via a right-sided suboccipital retrosigmoid approach. The patient's otalgia and mandibular pain disappeared after the operation. There were no additional neurological deficits. In conclusion, MVD is a safe and feasible option for patients with INN who fail to respond to adequate pharmacotherapy.

MicroRNA-9 inhibits vasculogenic mimicry of glioma cell lines by suppressing Stathmin expression.

The purpose of this study was to investigate the functions of microRNA-9, which is a tissue-specific microRNA in central nervous system, in the vasculogenic mimicry (VM) of glioma cell lines in vitro and in vivo. Glioma cell lines U87MG, U251 and SHG44 were transfected with microRNA-9 mimic, microRNA-9 inhibitor or scramble sequences. The amount of microRNA-9 and Stathmin (STMN1) mRNA was determined by quantitative real-time PCR, and the protein expression of STMN1 was determined by western blot. Cell proliferation and apoptosis were assessed. The interactions between the 3'UTR of STMN1 and miR-9 was determined by luciferase reporter assay. The VM capacity in vitro was evaluated using VM formation assay, and the rescue experiment of STMN1 was carried out in U251 cells. The in vivo experiment was applied with animal models implanted with U87MG cells.MicroRNA-9 mimic transfection reduced proliferation and increased apoptosis in glioma cell lines (p < 0.05). MicroRNA-9 mimic up-regulated STMN1 mRNA levels but reduced its protein levels (p < 0.05), and luciferase activity of STMN1 was suppressed by microRNA-9 mimic transfection (p < 0.05). Furthermore, microRNA-9 mimic transfection suppressed tumor volume growth, as well as VM both in vitro and in vivo. The cell viability and microtube density were upregulated in U251 cells after STMN1 up-regulation (p < 0.05). STMN1 is a target of microRNA-9, and microRNA-9 could modulate cell proliferation, VM and tumor volume growth through controlling STMN1 expression. MicroRNA-9 and its targets may represent a novel panel of molecules for the development of glioma treatment.

MicroRNA-489-3p aggravates neuronal apoptosis and oxidative stress after cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) mostly occurs in the treatment stage of ischemic diseases and aggravate brain tissue damage. Although studies have demonstrated that miR-489-3p is closely related to CIRI, the effects of miR-489-3p on neural function in CIRI have not been directly studied. The transient middle cerebral artery occlusion (tMCAO) model was established by suture method, and the corresponding plasmids that interfered with the expression of miR-489-3p or Sirtuin1 (SIRT1) were injected into the model mice, and the behavioral changes of the mice were observed. Then the concentration of serum neuronal injury markers and oxidative stress indices were examined. Next, the pathological conditions, neuronal loss and apoptosis of brain tissue were observed by hematoxylin-eosin staining, Nissl staining, and Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining. Finally, the hemoglobin content and cerebral edema in the mouse brain were determined. In addition, the expression levels of miR-489-3p and SIRT1 were detected by reverse transcription quantitative polymerase chain reaction or Western blot, and the targeting relationship between miR-489-3p and SIRT1 was verified by bioinformatics analysis and luciferase reporter assay. The experimental results found that in tMCAO mice, miR-489-3p in brain tissue was up-regulated and SIRT1 was down-regulated. Down-regulating miR-489-3p or up-regulating SIRT1 ameliorated behavioral dysfunction, neuronal damage and apoptosis, oxidative stress and brain histopathology. miR-489-3p targeted the regulation of SIRT1 expression, and down-regulating SIRT1 can reverse the protective effect of silenced miR-489-3p on brain injury. Taken together, by targeting SIRT1, elevated miR-489-3p aggravates CIRI-induced neuronal apoptosis and oxidative stress.

No association between EGF +61 A/G polymorphism and increased risk of glioma.

A single nucleotide polymorphism (SNP) of the epidermal growth factor (EGF) gene +61 A/G in the 5'-untranslated region has been reported to be associated with susceptibility to glioma. A case-control study (168 glioma patients and 194 normal controls) was conducted to elucidate its possible association with the risk of glioma in the Chinese population. Polymerase chain reaction-restriction fragment length polymorphism assay was used to analyze the EGF genotypes. The genotyping results were further confirmed by direct sequencing. The EGF +61A and +61G allele frequencies in the glioma group were 32.1% and 67.9%, respectively, while they were 30.4% and 69.6% in the healthy controls. Furthermore, the frequency of the A/A, A/G and G/G genotypes in glioma patients was 8.9%, 46.4%, and 44.7%, respectively, and 8.3%, 44.3%, and 47.4% in controls. There was no significant difference between patients and healthy controls. The EGF +61 A/G and +61 G/G genotypes were not significantly associated with risk of glioma compared with the A/A genotype. In addition, no significant association was observed between EGF polymorphism and different histological grades of glioma. These results indicate that the EGF +61 A/G polymorphism is not associated with susceptibility to glioma in the Chinese population. In addition, a literature review revealed a significantly higher rate of the A/A genotype in Caucasian compared with East Asian subjects. Such differences in genotype distribution between Caucasian and Asian people should be taken into account in future studies.

MicroRNA-9 enhances chemotherapy sensitivity of glioma to TMZ by suppressing TOPO II via the NF-κB signaling pathway.

Glioma is the most common primary tumor of the central nervous system (CNS) that develops chemotherapy resistance. The microRNA (miRNA) miR-9 is a tissue-specific miRNA of the CNS that may serve a key role in the modulation of chemotherapy sensitivity. The aim of the present study was to investigate the effect of miR-9 on glioma chemotherapy sensitivity by altering the expression of miR-9 in U251 glioma cells by viral transfection and subsequently treating with gradient concentrations of temozolomide (TMZ). Cell viability, apoptosis and the cell cycle were examined, and drug resistance genes were analyzed by western blotting. The role of nuclear factor κB (NF-κB) in this regulation was also examined. The results revealed that the susceptibility of glioma cells to TMZ was enhanced by miR-9 overexpression. When miR-9 and TMZ were applied together, the apoptotic rate and percentage of cells arrested at the G2/M stage were significantly higher compared with either treatment alone. Topoisomerase II expression was suppressed by miR-9 via the NF-κB signaling pathway, which may be responsible for the sensitization. The results of the present study suggested that miR-9 may be a potential target for glioma chemotherapy.

The IDH1 Mutation-Induced Oncometabolite, 2-Hydroxyglutarate, May Affect DNA Methylation and Expression of PD-L1 in Gliomas.

Background: Malignant gliomas are heterogeneous brain tumors with the potential for aggressive disease progression, as influenced by suppressive immunoediting. Given the success and enhanced potential of immune-checkpoint inhibitors in immunotherapy, we focused on the connections between genetic alterations affected by IDH1 mutations and immunological landscape changes and PDL-1 expression in gliomas. Methods: Paired surgically resected tumors from lower-grade gliomas (LGGs) and glioblastomas (GBM) were investigated, and a genetic analysis of patients' primary tumor samples culled from TCGA datasets was performed. Results: The results demonstrate that when compared with IDH1-mutant tumors, IDH1 wildtype tumors represent an immunosuppression landscape and elevated levels of PD-L1 expression. DNA hypo-methylation of the PD-L1 gene, as well as high gene and protein expressions, were observed in the wildtype tumors. We also found that quantitative levels of IDH1 mutant proteins were positively associated with recurrence-free survival (RFS). A key product of the IDH1 mutation (2-hydroxyglutarate) was found to transiently increase DNA methylation and suppress PD-L1 expression. Conclusions: IDH1 mutations impact the immune landscape of gliomas by affecting immune infiltrations and manipulating checkpoint ligand PD-L1 expression. Applications of immune checkpoint inhibitors may be beneficial for chemoradiation-insensitive IDH1-wildtype gliomas.

CD70, a novel target of CAR T-cell therapy for gliomas.

Background: Cancer immunotherapy represents a promising treatment approach for malignant gliomas but is hampered by the limited number of ubiquitously expressed tumor antigens and the profoundly immunosuppressive tumor microenvironment. We identified cluster of differentiation (CD)70 as a novel immunosuppressive ligand and glioma target. Methods: Normal tissues derived from 52 different organs and primary and recurrent low-grade gliomas (LGGs) and glioblastomas (GBMs) were thoroughly evaluated for CD70 gene and protein expression. The association between CD70 and patients' overall survival and its impact on T-cell death was also evaluated. Human and mouse CD70-specific chimeric antigen receptors (CARs) were tested respectively against human primary GBMs and murine glioma lines. The antitumor efficacies of these CARs were also examined in orthotopic xenograft and syngeneic models. Results: CD70 was not detected in peripheral and brain normal tissues but was constitutively overexpressed by isocitrate dehydrogenase (IDH) wild-type primary LGGs and GBMs in the mesenchymal subgroup and recurrent tumors. CD70 was also associated with poor survival in these subgroups, which may link to its direct involvement in glioma chemokine productions and selective induction of CD8+ T-cell death. To explore the potential for therapeutic targeting of this newly identified immunosuppressive axis in GBM tumors, we demonstrate that both human and mouse CD70-specific CAR T cells recognize primary CD70+ GBM tumors in vitro and mediate the regression of established GBM in xenograft and syngeneic models without illicit effect. Conclusion: These studies identify a previously uncharacterized and ubiquitously expressed immunosuppressive ligand CD70 in GBMs that also holds potential for serving as a novel CAR target for cancer immunotherapy in gliomas.

IDH1 R132H Mutation Is Accompanied with Malignant Progression of Paired Primary-Recurrent Astrocytic Tumours.

IDH1 R132H mutation is an important marker of survival in patients with gliomas. Although there are many changes of genes in tumour malignant progression, IDH1 R132H mutation status in glioma progression remained unclear. Here, an in-depth characterization of IDH1 R132H mutations were assessed by immunohistochemistry in 55 paired primary-recurrent astrocytomas tissues, including 5 paired primary pilocytic astrocytoma (pPA, WHO grade I), 35 paired primary low grade astrocytoma (pLGA, WHO grade II and III) and 15 paired primary high grade astrocytoma (pHGA/ Glioblastoma, WHO grade IV). Meanwhile, the DNA was isolated from paired samples, and PCR amplification was used for IDH1 exon4 sequencing. Nonparametric test, KM and Cox models were used to examine the statistical difference and survival function. We found that the percent of IDH1 R132H mutation was 68.6% (24/35) in pLGA group, but no IDH1 mutation was found in pPA and pHGA groups. Meanwhile, the results from immunohistochemistry and DNA sequencing showed that, compared with primary astrocytoma, there was no change of IDH1 status in recurrent astrocytoma whatever tumour pathological grade raise or indolent. The pPA group has the longest recurrence-free period (RFP) and overall survival (OS) in three groups (p<0.01), while the pHGA group has the shortest ones (p<0.01). In pLGA group, the IDH1 R132H mutation subgroup has longer RFP than IDH1 wild type subgroup (p<0.01), but the OS has no statistical difference between two subgroups (p>0.6). Additionally, IDH1 R132H mutation independently predicted a long RFP in patients with pLGA (HR 1.073, 95% CI 0.151-0.775, p<0.01).

Identifying survival-associated modules from the dysregulated triplet network in glioblastoma multiforme.

BACKGROUND: Long noncoding RNAs (lncRNAs) can act as competitive endogenous RNAs (ceRNAs) to compete with mRNAs for binding miroRNAs (miRNAs). The dysregulated triplets, composed by mRNAs, lncRNAs, and miRNAs, contributed to the development and progression of diseases, such as cancer. However, the roles played by triplet biomarkers are not fully understand in glioblastoma multiforme (GBM) patient survival. OBJECTIVES: Here, we constructed a differential triplet interaction network (TriNet) between GBM and normal tissues and identified GBM survival related triplets. METHODS: Four significantly dysregulated modules, enriched differentially expressed molecules, were identified by integrating affinity propagation method and hypergeometric method. Furthermore, knockdown of TP73-AS1 was implemented by siRNA and the expression of RFX1 was examined in U87 cells by qRT-PCR. The apoptosis of U87 cells was investigated using MTT assay and Acridine orange/Ethidium bromide (AO/EB) assay. RESULTS: We randomly split GBM samples into training and testing sets, and found that these four modules can robustly and significantly distinguish low- and high-survival patients in both two sets. By manually curated literatures for triplets mediated by core interactions, we found that members involved tumor invasion, proliferation, and migration. The dysregulated triplets may cause the poor survival of GBM patients. We finally experimentally verified that knockdown of TP73-AS1, an lncRNA of one triplet, could not only reduce the expression of RFX1, an mRNA of this triplet, but also induce apoptosis in U87 cells. CONCLUSIONS: These results can provide further insights to understand the functions of triplet biomarkers that associated with GBM prognosis.

CD4+ and Perivascular Foxp3+ T Cells in Glioma Correlate with Angiogenesis and Tumor Progression.

BACKGROUND: Angiogenesis and immune cell infiltration are key features of gliomas and their manipulation of the microenvironment, but their prognostic significance remains indeterminate. We evaluate the interconnection between tumor-infiltrating lymphocyte (TIL) and tumor blood-vasculatures in the context of glioma progression. METHODS: Paired tumor tissues of 44 patients from three tumor-recurrent groups: diffuse astrocytomas (DA) recurred as DA, DA recurred as glioblastomas (GBM), and GBM recurred as GBM were evaluated by genetic analysis, immunohistochemistry for tumor blood vessel density, TIL subsets, and clinical outcomes. These cells were geographically divided into perivascular and intratumoral TILs. Associations were examined between these TILs, CD34+ tumor blood vessels, and clinical outcomes. To determine key changes in TIL subsets, microarray data of 15-paired tumors from patients who failed antiangiogenic therapy- bevacizumab, and 16-paired tumors from chemo-naïve recurrent GBM were also evaluated and compared. RESULTS: Upon recurrence in primary gliomas, similar kinetic changes were found between tumor blood vessels and each TIL subset in all groups, but only CD4+ including Foxp3+ TILs, positively correlated with the density of tumor blood vessels. CD4 was the predominant T cell population based on the expression of gene-transcripts in primary GBMs, and increased activated CD4+ T cells were revealed in Bevacizumab-resistant recurrent tumors (not in chemo-naïve recurrent tumors). Among these TILs, 2/3 of them were found in the perivascular niche; Foxp3+ T cells in these niches not only correlated with the tumor vessels but were also an independent predictor of shortened recurrence-free survival (RFS) (HR = 4.199, 95% CI 1.522-11.584, p = 0.006). CONCLUSION: The minimal intratumoral T cell infiltration and low detection of CD8 transcripts expression in primary GBMs can potentially limit antitumor response. CD4+ and perivascular Foxp3+ TILs associate with tumor angiogenesis and tumor progression in glioma patients. Our results suggest that combining antiangiogenic agents with immunotherapeutic approaches may help improve the antitumor efficacy for patients with malignant gliomas.

S100B and ADMA in cerebral small vessel disease and cognitive dysfunction.

BACKGROUND: Glial cell activation and endothelial dysfunction are thought to contribute to the pathophysiology of cerebral small vessel disease (SVD). The purpose of the present study was to determine if levels of S100B, a protein highly expressed in glial cells, and asymmetric dimethylarginine (ADMA), which promotes endothelial dysfunction, are elevated in the serum of patients with SVD and correlate with their cognitive functioning. METHODS: The serum levels of S100B and ADMA were measured with enzyme-linked immunosorbent assays in 210 patients with SVD and 207 controls. Cognitive functioning was evaluated using the Montreal Cognitive Assessment. SVD lesions were categorized as isolated lacunar infarcts (ILI), multiple lacunar infarcts, leukoaraiosis (LA), and LA with cerebral atrophy using magnetic resonance imaging. RESULTS: SVD patients were significantly older, and more likely to have hypertension, diabetes, and heart disease, and smoke compared to controls (Ps<0.05). Plasma levels of S100B and ADMA were significantly higher in SVD patients (Ps<0.05), though only S100B was significant after adjusting for the confounding factors. Subtype analyses indicated that ADMA levels were differentially altered depending on lesion type, particularly in cases with ILI and LA (Ps<0.05). Compared with controls, SVD patients had significant cognitive impairment that was most profound in the cases with LA (all Ps<0.05). Levels of S100B and ADMA were significantly correlated with cognitive decline in patients with LA (P<0.05). CONCLUSION: S100B and ADMA are elevated in SVD, and are associated with cognitive impairment in patients with LA lesions.

Inhibition of SHP-2 promotes radiosensitivity in glioma.

As a phosphatase, SHP-2 has been identified to be involved in regulating several cell functions, including growth, division, adhesion and motility. Therefore, SHP­2 may affect the response of glioma to radiotherapy, such as via enhancing angiogenesis. The present study aimed to investigate the function of SHP­2, a protein tyrosine phosphatase, in the radiosensitivity of glioma. U251, U87 and SHG44 glioma cell lines were transfected with small interfering (si)RNA against SHP­2 and cell proliferation was assessed using a cell counting kit 8 assay, cell apoptosis was assessed by fluorescence­activated cell sorting and immunoblotting, cell invasion was determined by an invasion assay, and the vasculogenic mimicry capacity was assessed by a tube formation assay. SHP­2 siRNA transfection reduced the proliferation and increased apoptosis in the glioma cell lines. Downregulation of SHP­2 suppressed glioma cell invasion and vasculogenic mimicry. These results demonstrated that no significant difference was observed between glioma tissues and normal brain tissues, however, silencing of SHP­2 inhibited cell proliferation, invasion and vasculogenic mimicry in the glioma cell lines. SHP­2 may be a novel therapeutic target for glioma.

Stathmin expression in glioma-derived microvascular endothelial cells: a novel therapeutic target.

The purpose of this study was to investigate stathmin expression and its mechanisms of action in GDMEC. Microvascular endothelial cells were isolated from human gliomas (n=68) and normal brain specimans (n=20), and purified by magnetic beads coated with anti-CD105 antibody. The expression of stathmin mRNA and protein were detected by RT-PCR and western blotting, respectively. Stathmin expression was silenced by application of specific siRNA in high grade GDMEC. The proliferation, apoptosis and invasion behavior of GDMEC were investigated. The stathmin positive rate of endothelial cells in normal brain, grade I-II glioma and grade III-IV glioma was 20, 66 and 95.5%, respectively (P<0.05). When cells were treated with siRNA to silence stathmin, cell viability was reduced, the apoptosis rate increased and the migration of vascular endothelial cells was suppressed significantly (P<0.05). Down-regulation of stathmin suppressed neoangiogenesis of glioma and provides a potential target for glioma treatment.