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Nestin, a class IV intermediate filament protein, is generally considered as a putative marker of neural stem and progenitor cells in the central nervous system. Glioma is a common type of adult brain tumors, and glioblastoma (GBM) represents the most aggressive form of glioma. Here, we report that Nestin expression is significantly upregulated in human GBM, compared with other types of glioma. Nestin knockdown or deletion in U251 cells and tumor cells from GBM patients derived xenografts resulted in G2–M arrest, finally leading to apoptosis in tumor cells. Using proximity-dependent biotin identification method, we identified βII-tubulin as an interacting protein of Nestin in U251 cells. Nestin stabilized βII-tubulin in U251 cells through physical interaction. Knockdown of Nestin or βII-tubulin disrupted spindle morphology in tumor cells. Our studies further revealed that Nestin deficiency in U251 cells and GBM PDX cells repressed tumor growth upon transplantation. Finally, we found that Nestin deficiency sensitized GBM cells to microtubule-destabilizing drugs such as vinblastine and vincristine. Our studies demonstrate the essential functions and underlying mechanisms of Nestin in the growth and drug response of GBM cells.Implications:Through interaction with βII-tubulin, Nestin facilitates cell-cycle progression and spindle assembly of tumor cells in glioblastoma.

Supplementary Figure 1. Hedgehog pathway is not activated in U251 cells. Supplementary Figure 2. Colony formation from passaged U251 cells after Nestin knockdown. Supplementary Figure 3. GBM cells from PDX undergo apoptosis after Nestin knockdown. Supplementary Figure 4. Nestin deletion causes G2/M arrest and apoptosis in U251 cells. Supplementary Figure 5. Colony formation from U87 cells after transduction with Nestin shRNA. Supplementary Figure 6. No alterations in the mRNA expression of TUBB2A and TUBB2B in U251 cells after Nestin manipulation. Supplementary Figure 7. No alterations in the expression of Nestin mRNA and protein in U251 cells after βII-tubulin knockdown.

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Mounting evidence reveals that dysregulation of circular RNAs (circRNAs) is involved in the development of glioblastoma. Leucine-rich repeat-containing 4 (LRRC4) has been shown to suppress tumors in glioblastoma. However, whether LRRC4 can regulate the formation of circRNA is not yet understood. In this study, LRRC4 was found to interact with SAM68. LRRC4 promoted the generation of circCD44 by inhibiting the binding between SAM68 and CD44 pre-mRNA. Moreover, downregulated expression of circCD44 was found in glioblastoma multiforme (GBM) tissues and GBM primary cells. Re-expression of circCD44 significantly suppressed the proliferation, colony formation, and invasion of GBM cells and inhibited tumor growth in vivo. Mechanistically, circCD44 could regulate the expression of SMAD6 via sponging miR-326 and miR-330-5p involved in the progression of GBM. Thus, the LRRC4/SAM68/circCD44/miR-326/miR-330-5p/SMAD6 signaling axis could be a potential target for GBM treatment., Graphical abstract, Studies demonstrate that leucine-rich repeat-containing 4 promotes the generation of a novel suppressive circCD44 by restraining the binding between SAM68 and CD44 pre-mRNA, inhibits glioblastoma multiforme cell proliferation through the SAM68/circCD44/miR-326/miR-330-5p/SMAD6 signaling axis, and therefore provides a potential therapeutic target for glioblastoma.

Background Although major advances have been made in the histopathological diagnosis of high-grade astrocytoma (HGA), methods for effective and noninvasive diagnosis remain largely unknown. Exosomes can cross the blood–brain barrier and are readily accessible in human biofluids, making them promising biomarkers for HGA. Circular RNAs (circRNAs) have potential as tumor biomarkers owing to their stability, conservation, and tissue specificity. However, the landscape and characteristics of exosome circRNAs in HGA remain to be studied. Methods CircRNA deep sequencing and bioinformatics approaches were used to generate a circRNA profiling database and analyze the features of HGA cell circRNAs and HGA cell-derived exosome circRNAs. Exosome circRNA expression in the serum and tissues of healthy individuals and patients with HGA was detected using reverse transcription-quantitative PCR. Additionally, the receiver operating characteristic curve and overall survival curves were analyzed. Results By investigating the characteristics of HGA cell-derived exosome circRNAs and HGA cell circRNAs, we observed that exosomes were more likely to enrich short-exon and suppressor circRNAs than HGA cells. Moreover, a serum exosome circRNA panel including hsa_circ_0075828, hsa_circ_0003828, and hsa_circ_0002976 could be used to screen for HGA, whereas a good prognosis panel comprised high concentrations of hsa_circ_0005019, hsa_circ_0000880, hsa_circ_0051680, and hsa_circ_0006365. Conclusions This study revealed a comprehensive circRNA landscape in HGA exosomes and cells. The serum exosome circexosome circRNA panel and tissue circRNAs are potentially useful for HGA liquid biopsy and prognosis monitoring. Exosome circRNAs as novel targets should facilitate further biomarker discovery and aid in HGA diagnosis and therapy monitoring.

Nestin, a class IV intermediate filament protein, is generally considered as a putative marker of neural stem and progenitor cells in the central nervous system. Glioma is a common type of adult brain tumors, and glioblastoma (GBM) represents the most aggressive form of glioma. Here, we report that Nestin expression is significantly upregulated in human GBM, compared with other types of glioma. Nestin knockdown or deletion in U251 cells and tumor cells from GBM patients derived xenografts resulted in G2–M arrest, finally leading to apoptosis in tumor cells. Using proximity-dependent biotin identification method, we identified βII-tubulin as an interacting protein of Nestin in U251 cells. Nestin stabilized βII-tubulin in U251 cells through physical interaction. Knockdown of Nestin or βII-tubulin disrupted spindle morphology in tumor cells. Our studies further revealed that Nestin deficiency in U251 cells and GBM PDX cells repressed tumor growth upon transplantation. Finally, we found that Nestin deficiency sensitized GBM cells to microtubule-destabilizing drugs such as vinblastine and vincristine. Our studies demonstrate the essential functions and underlying mechanisms of Nestin in the growth and drug response of GBM cells.Implications:Through interaction with βII-tubulin, Nestin facilitates cell-cycle progression and spindle assembly of tumor cells in glioblastoma.

Investigating deuteron–deuteron (DD) fusion reactions in a plasma environment similar to the early stages of the Big Bang is an important topic in nuclear astrophysics. In this study, we experimentally investigated such reactions, using eight laser beams with the third harmonic impacting on a deuterated polyethylene target at the ShenGuang-II Upgrade laser facility. This work focused on the application of range-filter (RF) spectrometers, assembled from a 70 μm aluminum filter and two CR-39 nuclear track detectors, to measure the yields of primary DD-protons. Based on the track diameter calibration results of 3 MeV protons used to diagnose the tracks on the RF spectrometers, an approximate primary DD-proton yield of $$(8.5\pm 1.7)\times 10^6$$ was obtained, consistent with the yields from similar laser facilities worldwide. This indicates that the RF spectrometer is an effective way to measure primary DD-protons. However, due to the low yields of $${\hbox {D}}^{3}{\mathrm {He}}$$-protons and its small track diameter, CR-39 detectors were unable to distinguish it from the background spots. Using other accurate detectors may help to measure these rare events.

Ectopic epigenetic mechanisms play important roles in facilitating tumorigenesis. Here, we first demonstrated that ANKDD1A is a functional tumor suppressor gene, especially in the hypoxia microenvironment. ANKDD1A directly interacts with FIH1 and inhibits the transcriptional activity of HIF1α by upregulating FIH1. In addition, ANKDD1A decreases the half-life of HIF1α by upregulating FIH1, decreases glucose uptake and lactate production, inhibits glioblastoma multiforme (GBM) autophagy, and induces apoptosis in GBM cells under hypoxia. Moreover, ANKDD1A is highly frequently methylated in GBM. The tumor-specific methylation of ANKDD1A indicates that it could be used as a potential epigenetic biomarker as well as a possible therapeutic target.

Background Leucine rich repeat containing 4 (LRRC4), also known as netrin-G ligand-2 (NGL-2), belongs to the superfamily of LRR proteins and serves as a receptor for netrin-G2. LRRC4 regulates the formation of excitatory synapses and promotes axon differentiation. Mutations in LRRC4 occur in Autism Spectrum Disorder (ASD) and intellectual disability. Multiple sclerosis (MS) is a chronic autoimmune disease characterized by immune-mediated demyelination and neurodegeneration of the central nervous system (CNS). Here, we investigated the role of LRRC4 in the pathological process of experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS.Methods LRRC4 was detected in the CNS of EAE mice by the use of real-time PCR and western blotting. LRRC4 -/- mice were created and immunized with myelin oligodendrocyte glycoprotein peptide (MOG) 35–55 . Pathological changes in spinal cords of LRRC4 -/- and WT mice 15 days after immunization were examined by using hematoxylin and eosin (H&E), Luxol Fast Blue (LFB) staining and immunohistochemistry. The number of Th1/Th2/Th17/Treg cells in spleens and blood were measured with flow cytometry. Differential gene expression in the spinal cords from WT and LRRC4 -/- mice was analyzed by using RNA sequencing (RNA-seq). Adeno-associated virus (AAV) vectors were used to overexpress LRRC4 (AAV-LRRC4) and were injected into EAE mice to assess the therapeutic effect of AAV-LRRC4 ectopic expression on EAE.Results We discovered that the level of LRRC4 decreases in the spinal cords of the EAE mice. Deletion of LRRC4 accelerates infiltration of leukocytes into the spinal cords and disease exacerbation in vivo. We further showed that LRRC4 deletion disrupts the balance between Th1 cells and Treg cells and causes a shift toward Th1 cells and that the disrupted balance may be attributed to up-regulation of IL-6, IFN-γ and down-regulation of TNF-α in EAE mice. At a mechanistic level, we found that deficiency of LRRC4 induces elevated NF-κB p65 expression and does so by up-regulating Rab7b, while ectopic expression of LRRC4 alleviates the clinical symptoms of EAE mice and protects the CNS from immune damages.Conclusions We establish a critical role of LRRC4 in the progression of EAE and provide novel mechanistic insights into EAE development. Our findings also suggest that LRRC4 may be used as a potential target for therapeutic treatment of MS.

Temozolomide (TMZ) insensitivity and resistance are major causes of treatment failure and poor prognosis for GBM patients. Here, we identify LRRC4 as a novel autophagy inhibitor that restores the sensitivity of GBMs to TMZ. LRRC4 was associated with the DEPTOR/mTOR complex, and this interaction resulted in autophagy inhibition. Further investigation demonstrated that the PDZ binding domain of LRRC4 binds to the PDZ domain of DEPTOR. This binding decreases the half-life of DEPTOR via ubiquitination, thus inhibiting GBM cell autophagy and increasing the TMZ treatment response of GBM. Combined LRRC4 expression and TMZ treatment prolonged the survival of mice with tumour xenografts. Furthermore, the levels of LRRC4, DEPTOR and autophagy are clinically relevant for GBM, indicating that LRRC4 is likely to have significant potential as a therapeutic marker and target for TMZ treatment in glioma patients.

// Yunchao Li 1 , Chunhua Zhao 2 , Zhibin Yu 2 , Jiarui Chen 3 , Xiaoling She 4 , Peiyao Li 2 , Changhong Liu 2 , Yan Zhang 2 , Jianbo Feng 2 , Haijuan Fu 2 , Bing Wang 1 , Lei Kuang 1 , Lei Li 1 , Guohua Lv 1 , Minghua Wu 2 1 Department of Spinal Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China 2 Cancer Research Institute, School of Basic Medical Science, Central South University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Key Laboratory of Carcinogenesis, Ministry of Health, Changsha, Hunan, China 3 Xiangya Nursing School, Central South University, Changsha, Hunan, China 4 Pathology Department, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China Correspondence to: Guohua Lv, email: spinelv@163.com Minghua Wu, email: wuminghua554@aliyun.com Keywords: miR-381, osteosarcoma, LRRC4, chemosensitivity Received: April 09, 2016 Accepted: August 24, 2016 Published: September 06, 2016 ABSTRACT Osteosarcoma (OS) is the most common primary bone malignancy with a poor prognosis for all races and both sexes. In this study, we found that miR-381 is a positive prognosis factor for OS patients that OS patients with a low expression of miR-381 had a longer survival time after surgical intervention, and miR-381 expression promotes MG-63 cell proliferation and cell invasion ability. Our results also showed a strong negative correlation between the expression of miR-381 and LRRC4 (brain relative specific expression gene) in OS tissues. This demonstrated that LRRC4 is a direct target gene of miR-381, and suppressing the expression of miR-381 increases the sensitivity of OS cells to chemotherapeutic drugs through the LRRC4-mediated mTOR pathway. In summary, miR-381 is an important biomarker in directing therapeutic intervention and predicting prognosis in OS patients.

Background: Long non-coding RNAs (lncRNAs) play significant role in post-translational modifications of proteins, yet the importance of lncRNAs for sumoylation is unknown. Methods: RMST expression in glioma tissues and normal brain tissues was measured by RT-qPCR and in situ hybridization. The functional roles of RMST in astrocytomas were demonstrated by a series of in vitro experiments. The potential mechanisms of RMST for sumoylation was investigated by RNA immunoprecipitation,RNA pull-down, western blotting and co-immunoprecipitation assays. Findings: We first demonstrated the oncogenic activity of lncRNA RMST by inhibiting glioma cells mitophagy. We also first determined that RMST is an enhancer of FUS SUMOylation, especially boosting SUMO1 modification at K333. SUMOylation induced by RMST contributes to the interaction between FUS and hnRNPD and stabilized their expressionand cells mitophagy. Importantly, RMST as a highly expressed RNA in glioma is a better prognostic factor for glioma patients. Interpretation: our results demonstrated a previously unknown function of lncRNAs worked as an enhancer in FUS SUMOylation, and RMST will be a significant guide for the development of medications targeting gliomas. Funding: National Science Foundation of China;National Key Technology Research and Development program of the Ministry of Science and Technology of China;111 Project;Graduate Research and Innovation Projects of Central South University. Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: All of the protocols were reviewed and approved by the Joint Ethics Committee of the Central South University Health Authority and performed in accordance with national guidelines. Animal experiments were approved by the Animal Care and Use Committee of Central South University.

Background: Leucine-rich repeat containing 4(LRRC4) is low expression in GBM and our group first confirmed a new function of LRRC4 as a tumour suppressor for glioma. However, the underlying molecular mechanism and pathobiology of LRRC4 in autophagy and Temozolomide (TMZ) resistance remains largely unknown. Methods: TCGA date was used to examine the relationship between LRRC4 and autophagy element. LRRC4-overexpressing GBM cells were evaluated for autophagy level and TMZ resistance using cell-based functional assays and a mouse xenograft model. Furthermore, co-immunoprecipitation (CoIP) and mass spectrometry (MS) and were performed to identify the interaction partners of LRRC4. Findings: We confirmed the critical role of LRRC4 in regulation of autophagy. Ectopic expression of LRRC4 in GBM cells abrogates cells autophagy level and increases cell apoptosis when treated with TMZ. LRRC4 was associated with the DEPTOR/mTOR complex, and this interaction resulted in autophagy inhibition. Combined LRRC4 expression and TMZ treatment prolonged the survival of mice with tumour xenografts. Furthermore, the levels of LRRC4, DEPTOR and autophagy are clinically relevant for GBM. Interpretation: Our findings revealed that LRRC4 directly binds to DEPTOR and induces its degradation, thereby inhibiting cell autophagy. Moreover, autophagy inhibition increased the treatment efficacy of TMZ in glioma, and LRRC4-expressing cells underwent increased apoptosis with TMZ treatment, indicating that LRRC4 is likely to have significant potential as a therapeutic marker and target for TMZ treatment in glioma patients. Funding Statement: This works was supported by the National Natural Science Foundation of China (Grant No. 81874150) and Graduate Student Research Innovation Project in Hunan Province (2019zzts084). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All of the protocols were reviewed by the Joint Ethics Committee of the Central South University Health Authority and performed following national guidelines. All animal experiments were approved by the Animal Care and Use Committee of Central South University.

Tumor-associated macrophages (TAMs) constitute a major component of inflammatory cells in the glioblastoma multiforme (GBM) tumor microenvironment. TAMs have been implicated in GBM angiogenesis, invasion, local tumor recurrence, and immunosuppression. Coagulation factor X (FX) is a vitamin K-dependent plasma protein that plays a role in the regulation of blood coagulation. In this study, we first found that FX was highly expressed and positively correlated with TAM density in human GBM. FX exhibited a potent chemotactic capacity to recruit macrophages and promoted macrophages toward M2 subtype polarization, accelerating GBM growth. FX bound to extracellular signal-related kinase (ERK)1/2 and inhibited p-ERK1/2 in GBM cells. FX was secreted in the tumor microenvironment and increased the phosphorylation and activation of ERK1/2 and AKT in macrophages, which may have been responsible for the M2 subtype macrophage polarization. Moreover, although the lncRNA CASC2c has been verified to function as a miR-101 competing endogenous RNA (ceRNA) to promote miR-101 target genes in GBM cells, we first confirmed that CASC2c did not function as a miR-338-3p ceRNA to promote FX expression, and that FX was a target gene of miR-338-3p. CASC2c interacted with and reciprocally repressed miR-338-3p. Both CASC2c and miR-388-3p bound to FX and commonly inhibited its expression and secretion. CASC2c repressed M2 subtype macrophage polarization. Taken together, our findings revealed a novel mechanism highlighting CASC2c and FX as potential therapeutic targets to improve GBM patients by altering the GBM microenvironment.

Background Despite the overwhelming number of investigations on AKT, little is known about lncRNA on AKT regulation, especially in GBM cells. Methods RNA-binding protein immunoprecipitation assay (RIP) and RNA pulldown were used to confirm the binding of LINC00470 and fused in sarcoma (FUS). Confocal imaging, co-immunoprecipitation (Co-IP) and GST pulldown assays were used to detect the interaction between FUS and AKT. EdU assay, CCK-8 assay, and intracranial xenograft assays were performed to demonstrate the effect of LINC00470 on the malignant phenotype of GBM cells. RT-qPCR and Western blotting were performed to test the effect of LINC00470 on AKT and pAKT. Results In this study, we demonstrated that LINC00470 was a positive regulator for AKT activation in GBM. LINC00470 bound to FUS and AKT to form a ternary complex, anchoring FUS in the cytoplasm to increase AKT activity. Higher pAKT activated by LINC00470 inhibited ubiquitination of HK1, which affected glycolysis, and inhibited cell autophagy. Furthermore, higher LINC00470 expression was associated with GBM tumorigenesis and poor patient prognosis. Conclusions Our findings revealed a noncanonical AKT activation signaling pathway, i.e., LINC00470 directly interacts with FUS, serving as an AKT activator to promote GBM progression. LINC00470 has an important referential significance to evaluate the prognosis of patients. Electronic supplementary material The online version of this article (10.1186/s13045-018-0619-z) contains supplementary material, which is available to authorized users.

The epigenetics and genomics of glioblastoma (GBM) are complicated. Previous reports indicate that ELFN2 is widely distributed in the cerebral cortex neurons, striatum, and hippocampus cone and in granular cells. However, the function and mechanism of ELFN2, particularly in GBM, have rarely been explored. In this study, we identified ELFN2 as a new hypomethylation gene that acts as an oncogene in GBM. ELFN2 promoted cell autophagy by interacting with AurkA and eIF2α and inhibiting the activation of AurkA. We also demonstrated that aberrantly high ELFN2 expression is obtained due to hypomethylation of its promoter and abnormal miR-101 and LINC00470 expression in GBM. LINC00470 not only enhanced the expression of ELFN2 through adsorption of miR-101 but also affected the methylation level of ELFN2 by decreasing H3K27me3 occupancy. In addition, LINC00470 played a dominant role in the regulation of GBM cell autophagy, even though it upregulated ELFN2 expression. The results indicate that the combination of LINC00470 and ELFN2 has important significance for evaluating the prognosis of astrocytoma patients., Wu et al. identified ELFN2 as a hypomethylated gene that interacts with AurkA and eIF2α and promotes cell autophagy. Although LINC00470 mediates epigenetic regulation to contribute to drive ELFN2 expression, it plays a dominant suppressive role in autophagy. LINC00470 and ELFN2 have important significance for astrocytoma patients’ prognosis.

Effect of LINC00470 knockdown in GBM cells. RT-qPCR measured the expression of LINC00470 in GBM cell lines and primary GBM cells. Data presented as mean ± S.E.M. of three independent experiments. (DOCX 168 kb)

LINC00470 promoted GBM cell proliferation. CCK8 assay was performed to determine the viability of primary GBM cells. Primary GBM cells were transfected with si-NC and si-LINC00470, pcDNA3.1, and pcDNA3.1-LINC00470, respectively.*p

LINC00470, FUS, and AKT can form a ternary complex in U251 cells. The expression levels of LINC00470, AKT, and FUS were measured by RT-qPCR and Western blotting, respectively. Data presented as mean ± S.E.M. of three independent experiments. (DOCX 1262 kb)

The relationship between LINC00470, AKT, and p-AKT. A: RT-qPCR and Western blotting measured the expression of LINC00470 and AKT in GBM cell lines and primary GBM cells. Data presented as mean ± S.E.M. of three independent experiments. B: Western blotting measured the expression of AKT and p-AKT in GBM cell lines and primary GBM cells. Data showed positive correlation between the expression of LINC00470 and p-AKT in GBM. (DOCX 302 kb)

The associate between LINC00470, FUS, and AKT in U87 cells. A: the interaction of LINC00470 and FUS was detected through RIP assays in U87 cells. Data are presented as the mean ± S.E.M. of three independent experiments. **p

The expression of PI3K in GBM cells. The expression of PI3K was measured by Western blotting in GBM cells. (DOCX 204Â kb)

Supplementary figures S1â S13. (PDF 1864 kb)

Our previous report identified PR domain containing 16 (PRDM16), a member of the PR-domain gene family, as a new methylation associated gene in astrocytoma cells. This previous study also reported that miR-101 is a tumor suppressor in glioma. The present study confirms that PRDM16 is a hypomethylated gene that can be overexpressed in astrocytoma patients and demonstrates that the hypomethylation status of the PRDM16 promoter can predict poor prognoses for astrocytoma patients. The results reported herein show that PRDM16 was inhibited by miR-101 directly and also through epigenetic regulation. PRDM16 was confirmed as a new target of miR-101 and shown to be directly inhibited by miR-101. miR-101 also decreased the expression of PRDM16 by altering the methylation status of the PRDM16 promoter. miR-101 was associated with a decrease in the methylation-related histones H3K4me2 and H3K27me3 and an increase in H3K9me3 and H4K20me3 on the PRDM16 promoter. In addition, EZH2, EED and DNMT3A were identified as direct targets of miR-101, and miR-101 suppressed PRDM16 expression by targeting DNMT3A which decreases histone H3K27me3 and H3K4me2 at the PRDM16 core promoter. The results reported here demonstrate that miR-101 disrupted cellular mitochondrial function and induced cellular apoptosis via the mitochondrial pathway; for example, MMP and ATP levels decreased, while there was an increase in ADP/ATP ratios and ROS levels, levels of cleaved Caspase-9 and cleaved-PARP, the Bax/Bcl-2 ratios, and Smac release from the mitochondria to the cytoplasm. Knockdown of PRDM16 reversed the anti-apoptotic effect of miR-101 inhibition. In summary, miR-101 reversed the hypomethylation of the PRDM16 promoter which suppressed the expression of PRDM16, disrupted cellular mitochondrial function, and induced cellular apoptosis.