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Emx1IRES-Cre, D6-Cre and hGFAP-Cre are commonly used to conditionally manipulate gene expression or lineage tracing because of their specificity in the dorsal telencephalon during early neurogenesis as previously described. However, the spatiotemporal differences in Cre recombinase activity would lead to divergent phenotypes. Here, we compared the patterns of Cre activity in the early embryos among the three lines by mating with reporter mice. The activities of Emx1IRES-Cre, D6-Cre and hGFAP-Cre were observed in the dorsal telencephalon, starting from approximately embryonic day 9.5, 11.5 and 12.5, respectively. Although all the three lines have activity in radial glial cells, Emx1IRES-Cre fully covers the dorsal and medial telencephalon, including the archicortex and cortical hem. D6-Cre is highly restricted to the dorsal telencephalon with anterior-low to posterior-high gradients, partially covers the hippocampus, and absent in the cortical hem. Moreover, both Emx1IRES-Cre and hGFAP-Cre exhibit Cre activity outside the dorsal neocortex. Meanwhile, we used the three Cre lines to mediate Dicer knockout and observed inconsistent phenotypes, including discrepancies in radial glial cell number, survival and neurogenesis in the neocortex and hippocampus. Together we proved differences in Cre activity can perturb the resultant phenotypes, which aid researchers in appropriate experimental design.

Extracellular vesicles (EV), as the intercellular information transfer molecules which can regulate the tumor microenvironment, promote migration and tumor progression. Previous studies reported that EV from endothelial cells was used to guide the fate and survival of gliomas, but many researches focus on normal human endothelial cells (NhEC) rather than tumor-derived endothelial cells. Our laboratory isolated human endothelial cells from glioma issue (GhEC). We have previously demonstrated that EV from GhEC and NhEC, which both can promote glioma stem cells (GSC) proliferation and tumorsphere formation in vitro and tumourigenicity in vivo by the transfer of CD9. However, NhEC-EV or GhEC-EV could suppress glioma cells (GC) proliferation in vitro. It demonstrates the undifferentiated impact of EV. Here, we first compared GhEC-EV proteins with NhEC-EV (Screening criteria: GhEC-EV/NhEC-EV, FC > 1.5), and obtained 70 differential expression proteins, most of which were associated with invasion and migration. We found that GhEC or GhEC-EV preferred promoting GC migration than treating with NhEC or NhEC-EV. In terms of mechanism, we further revealed that EV-mediated transfer of MYO1C induced glioma cell LN229 migration. Knockdown of MYO1C in GhEC or GhEC-EV suppressed this effect. Overexpression of MYO1C promoted migration on the contrary. MYO1C was also detected in glioma cerebrospinal fluid (CSF), which is more suitable as a liquid biopsy biomarker and contributes to early diagnosis and monitoring in glioma. Our findings provide a new protein—MYO1C in EV to target tumor blood vessels, and bring a new point-cut to the treatment of gliomablastoma (GBM).

Glioma, the most common primary malignancy in the brain, has high recurrence and lethality rates, and thus, elucidation of the molecular mechanisms of this incurable disease is urgently needed. Poly-pyrimidine tract binding protein (PTBP1, also known as hnRNP I), an RNA-binding protein, has various mechanisms to promote gliomagenesis. However, the mechanisms regulating PTBP1 expression are unclear. Herein, we report a novel natural antisense noncoding RNA, PTB-AS, whose expression correlated positively with PTBP1 mRNA. We found that PTB-AS significantly promoted the proliferation and migration in vivo and in vitro of glioma cells. PTB-AS substantially increased the PTBP1 level by directly binding to its 3' UTR and stabilizing the mRNA. Furthermore, staphylococcal nuclease domain-containing 1 (SND1) dramatically increased the binding capacity between PTB-AS and PTBP1 mRNA. Mechanistically, PTB-AS could mask the binding site of miR-9 in the PTBP1-3' UTR; miR-9 negatively regulates PTBP1. To summarize, we revealed that PTB-AS, which maintains the PTBP1 level through extended base pairing to the PTBP1 3' UTR with the assistance of SND1, could significantly promote gliomagenesis.

Summary: Histone methylation is essential for regulating gene expression during organogenesis to maintain stem cells and execute a proper differentiation program for their descendants. Here we show that the COMPASS family histone methyltransferase co-factor ASH2L is required for maintaining neural progenitor cells (NPCs) and the production and positioning of projection neurons during neocortex development. Specifically, loss of Ash2l in NPCs results in malformation of the neocortex; the mutant neocortex has fewer neurons, which are also abnormal in composition and laminar position. Moreover, ASH2L loss impairs trimethylation of H3K4 and the transcriptional machinery specific for Wnt-β-catenin signaling, inhibiting the proliferation ability of NPCs at late stages of neurogenesis by disrupting S phase entry to inhibit cell cycle progression. Overexpressing β-catenin after ASH2L elimination rescues the proliferation deficiency. Therefore, our findings demonstrate that ASH2L is crucial for modulating Wnt signaling to maintain NPCs and generate a full complement of neurons during mammalian neocortex development. : Precise orchestration of gene expression dynamics is essential for stem cells to execute a proper differentiation program during organogenesis. Li et al. demonstrate that ASH2L, a core subunit of the COMPASS methyltransferase complex, is critical for maintaining neural progenitor cells and the generation of different neuronal types during mammalian neocortical development. Keywords: ASH2L, COMPASS, Wnt signaling pathway, corticogenesis, H3K4me3, cell cycle, neurogenesis

Gliomas account for approximately 80% of primary malignant tumors in the central nervous system. Despite aggressive therapy, the prognosis of patients remains extremely poor. Glioma stem cells (GSCs) which considered as the potential target of therapy for their crucial role in therapeutic resistance and tumor recurrence, are believed to be key factors for the disappointing outcome. Here, we took advantage of GSCs as the cell model to perform high-throughput drug screening and the old antibiotic, clofoctol, was identified as the most effective compound, showing reduction of colony-formation and induction of apoptosis of GSCs. Moreover, growth of tumors was inhibited obviously in vivo after clofoctol treatment especially in primary patient-derived xenografts (PDXs) and transgenic xenografts. The anticancer mechanisms demonstrated by analyzing related downstream genes and discovering the targeted binding protein revealed that clofoctol exhibited the inhibition of GSCs by upregulation of Kruppel-like factor 13 (KLF13), a tumor suppressor gene, through clofoctol's targeted binding protein, Upstream of N-ras (UNR). Collectively, these data demonstrated that induction of KLF13 expression suppressed growth of gliomas and provided a potential therapy for gliomas targeting GSCs. Importantly, our results also identified the RNA-binding protein UNR as a drug target.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has caused periodic infection outbreaks in children in the Asia–Pacific region. In order to describe the largely unknown life cycle of EV71, the molecular basis of its virus-host interactions must first be determined. The 5′ untranslated region of EV71 contains a cloverleaf-like structure and internal ribosomal entry site (IRES), which play an important role in transcription and translation of viral protein. We found that polypyrimidine tract-binding protein 1 (PTB) bound to the IRES of EV71. RNA recognition motifs 1 and 2 of PTB were responsible for its binding to the EV71 IRES. Moreover, PTB protein was shuttled from nucleus to cytoplasm after EV71 infection. Additionally, IRES activity and viral protein production were inhibited by PTB knockdown. These results suggest that PTB interacts with the EV71 IRES, and positively regulates viral protein translation. Electronic supplementary material The online version of this article (10.1007/s12250-019-00089-1) contains supplementary material, which is available to authorized users.

Nectin-like (Necl) molecules are Ca 2+ -independent Ig-like transmembrane cell adhesion molecules that participate in junctions between different cell types. The specific cell–cell adhesions mediated by Necl proteins are important in neural development and have been implicated in neurodegenerative diseases. Here, we present the crystal structure of the mouse Necl-4 full ectodomain and the structure of the heterophilic Necl ectodomain complex formed by the mNecl-4 and mNecl-1 ectodomains. We demonstrate that, while the ectodomain of mNecl-4 is monomeric, it forms a stable heterodimer with Ig1 of mNecl-1, with an affinity significantly higher than that observed for self-dimerization of the mNecl-1 ectodomain. We validated our structural characterizations by performing a surface plasmon resonance assay and an Fc fusion protein binding assay in mouse primary dorsal root ganglia neurites and Schwann cells and identified a selection of residues important for heterophilic interactions. Finally, we proposed a model of Necl binding specificity that involves an induced-fit conformational change at the dimerization interface.

Acidosis is a significant feature of the tumor microenvironment in glioma, and it is closely related to multiple biological functions of cancer stem cells. Here, we found that the self-renewal ability, the mitochondrial activity and ATP production were elevated in stem cell-like glioma cells (SLCs) under acidic microenvironment, which promoted and maintained the stemness of SLCs. Under acidosis, 25-hydroxy vitamin D3-24-hydroxylase (CYP24A1) was upregulated and catalyzed the fast degradation of 1α,25(OH)2D3. We further revealed that the active form of vitamin D (1α,25(OH)2D3) could inhibit the expression of stemness markers, attenuate acidosis-induced increase of self-renewal ability and mitochondrial respiration in stem cell-like glioma cells. Our study indicates that the acidosis–CYP24A1–vitamin D pathway may be a key regulator of the cancer stem cell phenotype in malignant glioma and point out the potential value for the utilization of vitamin D to target cancer stem cells and to restrain the growth of malignant glioma in the future.

Wnt/β-catenin signaling plays multiple important roles during mammalian brain development, and it regulates the proliferation and differentiation of neural progenitors in a context-dependent manner and affects neocortex layer formation. However, the specific role of Wnt/β-catenin in neuronal layer fate determination in the neocortex is still unclear. Here, we report that Zbed3, which is a positive regulator of Wnt/β-catenin signaling, colocalizes with β-catenin at the endfeet of radial glia in the ventricular zone of embryo mouse neocortex. Overexpression and knockdown of Zbed3 increased and decreased the activity of Wnt/β-catenin signaling in the neocortex, respectively. Interestingly, knockdown of Zbed3 in vivo could significantly shift neuronal fates from deep layers to upper layers but is not required for the proliferation and differentiation of neural progenitors. Overexpression of Zbed3 led to increased generation of deep-layer neurons without impairing cell cycle exit of neural progenitors. More importantly, knockdown of Zbed3 could effectively block the effects of the ectopic expression of stabilized β-catenin on neocortex layer formation. Hence, our results demonstrate that Zbed3 is indispensable for Wnt/β-catenin signaling regulating neuronal layer fates in the developing brain.

Glioma stem cells (GSCs) contribute to therapy resistance and poor outcomes for glioma patients. A significant feature of GSCs is their ability to grow in an acidic microenvironment. However, the mechanism underlying the rewiring of their metabolism in low pH remains elusive. Here, using metabolomics and metabolic flux approaches, we cultured GSCs at pH 6.8 and pH 7.4 and found that cells cultured in low pH exhibited increased de novo purine nucleotide biosynthesis activity. The overexpression of glucose-6-phosphate dehydrogenase, encoded by G6PD or H6PD, supports the metabolic dependency of GSCs on nucleotides when cultured under acidic conditions, by enhancing the pentose phosphate pathway (PPP). The high level of reduced glutathione (GSH) under acidic conditions also causes demand for the PPP to provide NADPH. Taken together, upregulation of G6PD/H6PD in the PPP plays an important role in acidic-driven purine metabolic reprogramming and confers a predilection toward glioma progression. Our findings indicate that targeting G6PD/H6PD, which are closely related to glioma patient survival, may serve as a promising therapeutic target for improved glioblastoma therapeutics. An integrated metabolomics and metabolic flux analysis, as well as considering microenvironment and cancer stem cells, provide a precise insight into understanding cancer metabolic reprogramming.

Enterovirus A71 (EV-A71) can cause hand, foot and mouth disease with neurological and systemic complications, most frequently affecting children and infants. We describe a cis-acting replication element (cre) with a conserved stem-loop structure within the EV-A71 2C-coding region. By site-directed mutagenesis and reverse genetics using the EV-A71 full-length genome and the EV-A71 replicon containing the firefly luciferase reporter gene in place of the P1 region, the stem-loop structure and the AAACA in the loop of the cre were confirmed to be required for the EV-A71 replication phenotype. EV-A71 genomes containing a mutation at the first or third A residue of AAACA could not be recovered. Insertion of a wild-type cre from EV-A71 or poliovirus in the 5’UTR led to successful recovery of the replication of nonviable mutants. Furthermore, the cre mutants showed lower binding capacity with the host cellular factor IGF2BP2, knockdown of which resulted in a significant decrease in EV-A71 production. All the available evidence shows the location independence but functional importance of the interaction of the cre with the cellular host for efficient production of EV-A71, contributing to the growing body of knowledge regarding picornavirus cres.

The eukaryotic cell cycle involves a highly orchestrated series of events in which the cellular genome is replicated during a synthesis (S) phase and each of the two resulting copies are segregated properly during mitosis (M). Host cell factor-1 (HCF-1) is a transcriptional co-regulator that is essential for and has been implicated in basic cellular processes, such as transcriptional regulation and cell cycle progression. Although a series of HCF-1 transcriptional targets have been identified, few functional clues have been provided, especially for chromosome segregation. Our results showed that HCF-1 activated CDC42 expression by binding to the −881 to −575 region upstream of the CDC42 transcription start site, and the regulation of CDC42 expression by HCF-1 was correlated with cell cycle progression. The overexpression of a spontaneously cycling and constitutively active CDC42 mutant (CDC42F28L) rescued G1 phase delay and multinucleate defects in mitosis upon the loss of HCF-1. Therefore, these results establish that HCF-1 ensures proper cell cycle progression by regulating the expression of CDC42, which indicates a possible mechanism of cell cycle coordination and the regulation mode of typical Rho GTPases.

RNA-binding proteins (RBPs) play important roles in many cancer types. However, RBPs have not been thoroughly and systematically studied in gliomas. Global analysis of the functional impact of RBPs will provide a better understanding of gliomagenesis and new insights into glioma therapy. In this study, we integrated a list of the human RBPs from six sources&mdash, Gerstberger, SONAR, Gene Ontology project, Poly(A) binding protein, CARIC, and XRNAX&mdash, which covered 4127 proteins with RNA-binding activity. The RNA sequencing data were downloaded from The Cancer Genome Atlas (TCGA) (n = 699) and Chinese Glioma Genome Atlas (CGGA) (n = 325 + 693). We examined the differentially expressed genes (DEGs) using the R package DESeq2, and constructed a weighted gene co-expression network analysis (WGCNA) of RBPs. Furthermore, survival analysis was also performed based on the univariate and multivariate Cox proportional hazards regression models. In the WGCNA analysis, we identified a key module involved in the overall survival (OS) of glioblastomas. Survival analysis revealed eight RBPs (PTRF, FNDC3B, SLC25A43, ZC3H12A, LRRFIP1, HSP90B1, HSPA5, and BNC2) are significantly associated with the survival of glioblastoma patients. Another 693 patients within the CGGA database were used to validate the findings. Additionally, 3564 RBPs were classified into canonical and non-canonical RBPs depending on the domains that they contain, and non-canonical RBPs account for the majority (72.95%). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that some non-canonical RBPs may have functions in glioma. Finally, we found that the knockdown of non-canonical RBPs, PTRF, or FNDC3B can alone significantly inhibit the proliferation of LN229 and U251 cells. Simultaneously, RNA Immunoprecipitation (RIP) analysis indicated that PTRF may regulate cell growth and death- related pathways to maintain tumor cell growth. In conclusion, our findings presented an integrated view to assess the potential death risks of glioblastoma at a molecular level, based on the expression of RBPs. More importantly, we identified non-canonical RNA-binding proteins PTRF and FNDC3B, showing them to be potential prognostic biomarkers for glioblastoma.

Now, numerous exciting findings have been found that long noncoding RNAs (lncRNAs) play a vital role in cancer malignant progression. However, their potential involvement in glioma is not well understood. Here, we performed a high-throughput microarray to detect the lncRNA expression profiles between glioma cell lines and normal astrocyte cell lines. HOXD-AS2 was increased in glioma cells and it was associated with glioma grade and poor prognosis. Loss of HOXD-AS2 can inhibit glioma cell growth by inducing cell-cycle G1 arrest in vitro. The proliferation of glioma was inhibited followed by knocking down the expression of HOXD-AS2 not only in subcutaneous injection model but also in orthotopic implantation model. These findings indicate that HOXD-AS2 promotes the glioma progression and may serve as a potential target for glioma diagnosis and therapy.

Differentiation status significantly affects the properties of malignant glioma cells, with non-stem cells inducing tumor enlargement and stem-like cells driving tumor initiation and treatment resistance. It is not completely understood how the same protein can have a distinct role in these cell populations. Here, we report that four and a half LIM domain protein 3 (FHL3) has an inhibitory effect on proliferation in non-stem glioma cells and a non-proliferative effect in glioma stem cells (GSCs). In GSCs, we show that FHL3 interacts with the Smad2/3 protein complex at the SOX4 promoter region, inhibits SOX4 transcriptional activity by recruiting PPM1A phosphatase to Smad2/3, and then suppresses GSC tumor sphere formation and self-renewal in vitro and in vivo via downregulation of SOX2 expression. Altogether, these findings highlight the role of FHL3 as a stemness-suppressor in regulation of the Smad2/3-SOX4-SOX2 axis in glioma.

To investigate the association of the Nectin/Necl family genes with the risk of developing NMOSD.Whole-exome sequencing was performed on two familial NMOSD cases and two unaffected family members. Additionally, 106 patients with sporadic NMOSD and 212 healthy controls (HCs) underwent screening for mutant Necl2. Finally, the molecular weight and cellular localization of mutant NECL2 was examined in transfected HeLa cells.We identified a novel deletion mutation in Necl2 (c.1052_1060delCCACCACCA; p. Thr351_Thr353del), which was associated with disease manifestation in the NMOSD familial cases. The frequency at which the mutation occurred in patients with sporadic NMOSD was significantly higher than for HCs (5.7% and 0, respectively; p0.01). The mutation was located in the extracellular domain close to the transmembrane region, at a point in the protein sequence characterized by threonine enrichment. The mutant NECL2 had a lower molecular weight and exhibited defective trafficking to the cell surface.Our results suggest that the Necl2 mutation identified herein may be associated with the risk of developing NMOSD. Furthermore, mutated NECL2 may play a role in the pathogenesis of the disease, potentially through its roles in axonal regeneration and/or via neuron-glia interactions that are relevant to myelination.

T-UCRs, a class of long non-coding RNAs that are transcribed from ultra-conserved regions (UCRs), might play an important role in development and diseases. However, the amount of T-UCRs that are conservatively expressed in the developing nervous systems of mice, monkeys and humans is still unknown. In this study, we screened the RNA sequence signals of 481 identified UCRs in an E14.5 mouse brain from the ENCODE database and found 76 UCRs that may be transcribed into T-UCRs. To verify the expression of these potential T-UCRs, we used an RT-PCR experiment and identified that 60 T-UCRs can be expressed in the E14.5 mouse brain. Furthermore, we detected the expression conservation of 76 potential T-UCRs in two comparisons: postnatal day 0 brains of a mouse and a rhesus monkey and neural stem cells of mouse and human by RT-PCR experimentation. It was found that up to 65% of these T-UCRs were expressed in mouse, rhesus monkey and human nervous systems. Next, by testing the spatiotemporal expression pattern of these T-UCRs expressed in mouse, rhesus monkey and human nervous systems, we found that approximately 30% of the T-UCRs showed a relatively high and dynamical expression during mouse brain development. Finally, through biological process and molecular function gene ontology analysis of the host genes of intronic or exonic-antisense T-UCRs, it was discovered that most of the genes were involved in RNA splicing or RNA binding. These results suggest that T-UCRs are likely to participate in nervous system development through RNA processing.

Gliomas are the most common and lethal primary malignant brain tumors. Recent studies implicate an important role for a rare population of glioma stem cells (GSCs) in glioma maintenance and recurrence. New therapeutic strategies are desperately needed requiring insights into the biological and molecular mechanisms underlying the self-renewal and differentiation of GSCs. We now investigate the metabolic signatures of three glioma cell lines with different stemness using a liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach. Cellular metabolites differentially expressed in U87MG stem-like cells (SLCs) relative to U87 malignant glioma cells (GCs) and U87MG stem-like cell differentiation cells (SLCDCs) were identified. The specific and significant alterations including nucleotide metabolism, glycerophospholipid metabolism, glutathione metabolism, carnitine metabolism and tryptophan metabolism were characterized. Cell function assays were further used to evaluate the self-renewal ability of SLCs treated with differential metabolites, indicating that these metabolites are involved in the maintenance of stemness. The results provide valuable information on the association of the significantly altered metabolites and metabolic pathways with SLC self-renewal and differentiation.

During cortical development, neuronal migration is one of the most important steps for normal cortical formation and function, and defects in this process cause many brain diseases. However, the molecular mechanisms underlying this process remain largely unknown. In this study, we found that miR-129-5p and miR-129-3p were expressed in both neural progenitor cells and cortical neurons in the developing murine cortex. Moreover, abnormal miR-129 expression could block radial migration of both the deeper layer and upper layer neurons, and impair the multipolar to bipolar transition. However, antagomir-mediated inhibition resulted in overmigration of neurons. In addition, we showed that Fragile X Mental Retardation gene 1 (Fmr1), which is mutated in the autism spectrum disorder fragile X syndrome, is an important regulatory target for miR-129-5p. Furthermore, Fmr1 loss-of-function and gain-of-function experiments showed opposite effects on miR-129 regulation of neuronal migration, and restoring Fmr1 expression could counteract the deleterious effect of miR-129 on neuronal migration. Taken together, our results suggest that miR-129-5p could modulate the expression of fragile X mental retardation 1 protein (FMRP) to ensure normal neuron positioning in the developing cerebral cortex.

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P

Cell fate specification in neural progenitor cells (NPCs) is orchestrated via extrinsic and intrinsic molecular programs, and histone methylation in these decisions has been ascribed to a crucial function regulating gene expression. Here, we show that the COMPASS family histone methyltransferase co-factor ASH2L is required in NPCs proliferation and upper layer cortical projection neurons production and position. Deletion of Ash2l impairs trimethylation of H3K4 and transcriptional machinery specifically for subsets of Wnt-β-catenin signalling, disrupting their transcription and consequently inhibiting the proliferation ability of NPCs in late stages of neurogenesis. Consistently, Ash2l conditional mutants exhibit thinning neocortex with reduced upper layer neurons and altered neuronal position. Moreover, overexpressing β-catenin after Ash2l elimination or knockdown can rescue the proliferation deficiency of NPCs both in vivo and in vitro. These results demonstrate an essential and highly specific role for Ash2l in controlling NPCs proliferation and late-born neurons lamination in corticogenesis via transcriptionally regulating Wnt-β-catenin signalling, and provide clues to how the COMPASS family epigenetic factors coordinate cell fate determination during cortex development.

MicroRNAs (miRNAs) control various biological processes by inducing translational repression and transcript degradation of the target genes. In mammalian development, knowledge of the timing and expression pattern of each miRNA is important to determine and predict its function in vivo. So far, no systematic analyses of the spatiotemporal expression pattern of miRNAs during mammalian neurodevelopment have been performed. Here, we isolated total RNAs from the embryonic dorsal forebrain of mice at different developmental stages and subjected these RNAs to microarray analyses. We selected 279 miRNAs that exhibited high signal intensities or ascending or descending expression dynamics. To ascertain the expression patterns of these miRNAs, we used locked nucleic acid (LNA)-modified miRNA probes in in situ hybridization experiments. Multiple miRNAs exhibited spatially restricted/enriched expression in anatomically distinct regions or in specific neuron subtypes in the embryonic brain and spinal cord, such as in the ventricular area, the striatum (and other basal ganglia), hypothalamus, choroid plexus, and the peripheral nervous system. These findings provide new insights into the expression and function of miRNAs during the development of the nervous system and could be used as a resource to facilitate studies in neurodevelopment.

The perivascular niche in glioma is critical for the maintenance of glioma stem cells (GSCs), and tumour-endothelial cell (EC) communication impacts tumourigenesis in ways that are incompletely understood. Here, we show that glioma-associated human endothelial cells (GhECs), a main component of the perivascular niche, release extracellular vesicles (EVs) that increase GSC proliferation and tumour-sphere formation. GSCs treated with GhEC-EVs create a significantly greater tumour burden than do untreated GSCs in orthotopic xenografts. Mechanistic, analysis of EVs content identified CD9 as a mediator of the effects on GSCs. CD9 can activate the BMX/STAT3 signalling pathway in GSCs. Our results illuminate the tumour-supporting role of ECs by identifying that EC-derived EVs transfer of CD9 during intercellular communication, thereby enhancing the aggressiveness of glioblastoma by specifically maintaining GSCs.

Brain-enriched miR-128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR-128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR-128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR-128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR-128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR-128 were screened and laid the foundation for research into the miR-128 regulation network.

The accurate generation of an appropriate number of different neuronal and glial subtypes is fundamental to normal brain functions and requires tightly orchestrated spatial and temporal developmental programmes to maintain the balance between the proliferation and the differentiation of neural progenitor cells. However, the molecular mechanism governing this process has not been fully elucidated. Here, we found that miR-214-3p was highly expressed in neural progenitor cells and dynamically regulated during neocortical development. Moreover, our in vivo and in vitro studies showed that miR-214 inhibited self-renewal of neural progenitor cells and promoted neurogenesis. In addition, after target screening, we identified miR-214 targets including Quaking (Qki) by binding the 3′- untranslated region (3′-UTR) of the Qki mRNA, which was specifically expressed in the progenitor cells of the proliferative ventricular zone as 3 Qki isoforms. Furthermore, overexpression and knockdown of Qki showed that the different isoforms of Qki had different functions in the regulation of neural progenitor cells differentiation. Moreover, overexpression of Qki could counteract the function of miR-214 in neurogenesis. Our results revealed that miR-214 maintains the balance between neural progenitor/stem cell proliferation and differentiation together with Quaking, its target gene.

Summary The precisely timed generation of different neuronal types is a hallmark of development from invertebrates to vertebrates. In the developing mammalian neocortex, neural stem cells change competence over time to sequentially produce six layers of functionally distinct neurons. Here, we report that microRNAs (miRNAs) are dispensable for stem-cell self-renewal and neuron production but essential for timing neocortical layer formation and specifying laminar fates. Specifically, as neurogenesis progresses, stem cells reduce miR-128 expression and miR-9 activity but steadily increase let-7 expression, whereas neurons initially maintain the differences in miRNA expression present at birth. Moreover, miR-128, miR-9, and let-7 are functionally distinct; capable of specifying neurons for layer VI and layer V and layers IV, III, and II, respectively; and transiently altering their relative levels of expression can modulate stem-cell competence in a neurogenic-stage-specific manner to shift neuron production between earlier-born and later-born fates, partly by temporally regulating a neurogenesis program involving Hmga2.

It has been reported that ubiquitin-conjugating enzyme 9 (Ubc9),the unique enzyme2 in the sumoylation pathway, is up-regulatedin many cancers. However, the expression and regulation ofUBC9 in glioma remains unknown. In this study, we found thatUbc9 was up-regulated in glioma tissues and cell lines comparedto a normal control. UBC9 knockdown by small interfering RNA(siRNA) affected cell proliferation and apoptosis in T98G cells.Further experiments revealed that microRNA (miR)-214 directlytargeted the 3' untranslated region (UTR) of UBC9 and that therewas an inverse relationship between the expression levels ofmiR-214 and UBC9 protein in glioma tissues and cells. miR-214overexpression suppressed the endogenous UBC9 protein andaffected T98G cell proliferation. These findings suggest thatmiR-214 reduction facilitates UBC9 expression and is involvedin the regulation of glioma cell proliferation

Protein arginine methyltransferase 1 (PRMT1), a type-I argininemethyltransferase, has been implicated in diverse cellularevents. We have focused on the role of PRMT1 in gliomagenesis.In this study, we showed that PRMT1 expression wasup-regulated in glioma tissues and cell lines compared withnormal brain tissues. The knock-down of PRMT1 resulted in anarrest in the G1-S phase of the cell cycle, proliferation inhibitionand apoptosis induction in four glioma cell lines(T98G, U87MG, U251, and A172). Moreover, an in vivo studyconfirmed that the tumor growth in nude mouse xenografts wassignificantly decreased in the RNAi-PRMT1 group. Additionally,we found that the level of the asymmetric dimethylatedmodification of H4R3, a substrate of PRMT1, was higher inglioma cells than in normal brain tissues and decreased afterPRMT1 knock-down. Our data suggest a potential role forPRMT1 as a novel biomarker of and therapeutic target ingliomas.

The insulin receptor substrate (IRS) family plays important roles in cellular growth, signaling, and survival in the brain. We identified IRS6/Dok-5, a member of the IRS family, also expressed in heart. Dok-5 expression level significantly increased during cardiomyocyte differentiation of P19CL6 cells. To understand the mechanism of Dok-5 gene expression and regulation during cardiomyocyte differentiation, we first mapped the transcription start site of the mouse Dok-5 gene and characterized its promoter regions. Truncation and mutation analysis of the Dok-5 promoter identified the forkhead binding element responsible for the repression of Dok-5 promoter activation. The co-localization of FOXO3a and Dok-5 in the mouse heart allows FOXO3a to be a transcriptional regulator of Dok-5. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed that FOXO3a could bind to the Dok-5 promoter, accompanied by FOXO3a translocation from the nucleus to cytoplasm. FOXO3a overexpression could inhibit Dok-5 promoter activity. Silencing FOXO3a expression by siRNA upregulated the expression of Dok-5 and enhanced cardiomyocyte differentiation. Moreover, Dok-5 siRNA attenuated cardiomyocyte differentiation. Our results provide the first evidence that FOXO3a, the PI3K/PKB downstream substrate, acts as a transcriptional repressor to inhibit the expression of Dok-5. Dok-5 is involved in cardiomyocyte differentiation by a PI3K/PKB/FOXO3a signaling pathway.

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue-specific immunoglobulin-like cell–cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re-expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at −271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor-Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co-immunoprecipitation (co-IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation. © 2008 Wiley-Liss, Inc.