Expression and regulatory roles of lncRNAs in G-CIMP-low vs G-CIMP-high Glioma: an in-silico analysis

BACKGROUND: Clinically relevant glioma subtypes, such as the glioma-CpG island methylator phenotype (G-CIMP), have been defined by epigenetics. In this study, the role of long non-coding RNAs in association with the poor-prognosis G-CMIP-low phenotype and the good-prognosis G-CMIP-high phenotype was investigated. Functional associations of lncRNAs with mRNAs and miRNAs were examined to hypothesize influencing factors of the aggressive phenotype. METHODS: RNA-seq data on 250 samples from TCGA\u27s Pan-Glioma study, quantified for lncRNA and mRNAs (GENCODE v28), were analyzed for differential expression between G-CIMP-low and G-CIMP-high phenotypes. Functional interpretation of the differential lncRNAs was performed by Ingenuity Pathway Analysis. Spearman rank order correlation estimates between lncRNA, miRNA, and mRNA nominated differential lncRNA with a likely miRNA sponge function. RESULTS: We identified 4371 differentially expressed features (mRNA = 3705; lncRNA = 666; FDR ≤ 5%). From these, the protein-coding gene TP53 was identified as an upstream regulator of differential lncRNAs PANDAR and PVT1 (p = 0.0237) and enrichment was detected in the development of carcinoma (p = 0.0176). Two lncRNAs (HCG11, PART1) were positively correlated with 342 mRNAs, and their correlation estimates diminish after adjusting for either of the target miRNAs: hsa-miR-490-3p, hsa-miR-129-5p. This suggests a likely sponge function for HCG11 and PART1. CONCLUSIONS: These findings identify differential lncRNAs with oncogenic features that are associated with G-CIMP phenotypes. Further investigation with controlled experiments is needed to confirm the molecular relationships

Insulin-like Growth Factor Binding Protein 7 Mediates Glioma Cell Growth and Migration

Insulin-like growth factor binding protein 7 (IGFBP-7) is the only member of the IGFBP superfamily that binds strongly to insulin, suggesting that IGFBP-7 may have different functions from other IGFBPs. Unlike other IGFBPs, the expression and functions of IGFBP-7 in glioma tumors have not been reported. Using cDNA microarray analysis, we found that expression of IGFBP-7 correlated with the grade of glioma tumors and the overall patient survival. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. We used RNAi to examine the role of IGFBP-7 in glioma cells, inhibiting IGFBP-7 expression by short interfering RNA transfection. Cell proliferation was suppressed after IGFBP-7 expression was inhibited for 5 days, and glioma cell growth was stimulated consistently by the addition of recombinant IGFBP-7 protein. Moreover, glioma cell migration was attenuated by IGFBP-7 depletion but enhanced by IGFBP-7 overexpression and addition. Overexpression of AKT1 in IGFBP-7-overxpressed cells attenuated the IGFBP-7-promoted migration and further enhanced inhibition of IGFBP-7 depletion on the migration. Phosphorylation of AKT and Erk1/2 was also inversely regulated by IGFBP-7 expression. These two factors together suggest that IGFBP-7 can regulate glioma cell migration through the AKT-ERK pathway, thereby playing an important role in glioma growth and migration

Differential Regulation of Bcl-2 and Bax Expression in Cells Infected with Virulent and Nonvirulent Strains of Sindbis Virus

AbstractSindbis virus is an alphavirus that infects cells in either lytic or persistent infection. In this study we examined the effects of Sindbis virus on cell apoptosis and on the expression of Bcl-2 and Bax. Of the two strains studied, SVA and SVNI, only the neurovirulent strain, SVNI, induced apoptosis of astrocytes and PC-12 cells. SVA, which infects cells in a persistent manner, induced up-regulation of bcl-2 mRNA and Bcl-2 protein, whereas SVNI induced an increase in Bax levels. Our results indicate a differential regulation of Bcl2 and Bax expression by SVA and SVNI, which may be associated with the apoptotic potential of the viruses

sPIF promotes myoblast differentiation and utrophin expression while inhibiting fibrosis in Duchenne muscular dystrophy via the H19/miR-675/let-7 and miR-21 pathways

Duchenne muscular dystrophy (DMD) is a progressive, lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Loss of dystrophin leads to muscle fiber damage and impairment of satellite cell asymmetric division, which are essential for muscle regeneration. These processes ultimately result in muscle wasting and the replacement of the degenerating muscles by fibrogenic cells, a process that leads to the generation of fibrotic tissues. Preimplantation factor (PIF) is an evolutionary conserved 15-amino acid peptide secreted by viable mammalian embryos. Synthetic PIF (sPIF) reproduces the protective/regenerative effects of the endogenous peptide in immune disorders and transplantation models. In this study, we demonstrated that sPIF treatment promoted mouse and human myoblast differentiation and inhibited the expression of collagen 1A1, collagen 1A2, and TGF-β in DMD patient-derived myoblasts. Additionally, sPIF increased the expression of utrophin, a homolog of dystrophin protein. sPIF effects were mediated via the upregulation of lncRNA H19 and miR-675 and downregulation of let-7. sPIF also inhibited the expression of miR-21, a major fibrosis regulator. The administration of sPIF in mdx mice significantly decreased serum creatine kinase and collagen I and collagen IV expression in the diaphragm, whereas it increased utrophin expression in the diaphragm, heart and quadriceps muscles. In conclusion, sPIF promoted the differentiation of DMD myoblasts, increased utrophin expression via the H19/miRNA-675/let-7 pathway, and reduced muscle fibrosis possibly via the upregulation of miR-675 and inhibition of miR-21 expression. These findings strongly support pursuing sPIF as a potential therapeutic agent for DMD. Moreover, the completion of an sPIF phase I safety trial will further promote the use of sPIF for the treatment of muscular dystrophies

Neuron-glia crosstalk mediate the neurotoxic effects of ketamine via extracellular vesicles

Background: General anesthetics (GA) are associated with neurodevelopmental abnormalities including cell death, cognitive and behavioral changes. There is now powerful evidence for non-cell autonomous mechanisms in almost every pathological condition in the brain, especially relevant to glial cells, mainly astrocytes and microglia, that exhibit structural and functional contacts with neurons. These interactions were recently reported to occur via the secretion of extracellular vesicles (EVs). Here, we employed primary human neural cells to analyze ketamine effects focusing on the functions of glial cells and their polarization/differentiation state. We also explored the roles of extracellular vesicles (EVs) and different components of the BDNF pathway. Methods: Ketamine effects were analyzed on human neuronal and glial cell proliferation and apoptosis and astrocytic (A1/A2 ) and microglial (M1/M2) cell activation were analyzed. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell co-cultures. The role of the brainderived neurotrophic factor (BDNF) pathway, was analyzed using RT-PCR, ELISA western blot and gene silencing. EVs secreted by ketamine-treated cells were isolated, characterized and analyzed for their effects in neuron-glia cell interactions. Data were analyzed using analysis of variance or a Student\u27s t test with correction for data sets with unequal variances. Results: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted the expression of proinflammatory astrocytes (A1) and microglia (M1) phenotypes. Astrocytes and microglia enhanced the neurotoxic effects of ketamine on neuronal cells, whereas neurons increased oligodendrocyte cell death. Ketamine modulated different components in the BDNF pathway: decreasing BDNF secretion in neurons and astrocytes while increasing the expression of p75 in neurons and oligodendrocytes. In addition, ketamine treatment increased the lncRNA BDNF-AS levels and the secretion of pro-BDNF secretion. We found an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death by delivering BDNF-AS. Conclusions: Ketamine neurotoxicity involves both autonomous and non-cell autonomous mechanisms andomponents of the BDNF pathway expressed by neurons and glial cells represent major regulators of ketamine effects. We demonstrated for the first time a role of EVs as important mediators of ketamine effects by the delivery of specific non-coding RNAs. These results may contribute to a better understanding of cellular and molecular mechanisms underlying ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact

Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome

Inactivating mutations in the Methyl-CpG Binding Protein 2 (MECP2) gene are the main cause of Rett syndrome (RTT). Despite extensive research into MECP2 function, no treatments for RTT are currently available. Here, we used an evolutionary genomics approach to construct an unbiased MECP2 gene network, using 1028 eukaryotic genomes to prioritize proteins with strong co-evolutionary signatures with MECP2. Focusing on proteins targeted by FDA-approved drugs led to three promising targets, two of which were previously linked to MECP2 function (IRAK, KEAP1) and one that was not (EPOR). The drugs targeting these three proteins (Pacritinib, DMF, and EPO) were able to rescue different phenotypes of MECP2 inactivation in cultured human neural cell types, and appeared to converge on Nuclear Factor Kappa B (NF-κB) signaling in inflammation. This study highlights the potential of comparative genomics to accelerate drug discovery, and yields potential new avenues for the treatment of RTT

Specific Compositions of Cannabis sativa Compounds Have Cytotoxic Activity and Inhibit Motility and Colony Formation of Human Glioblastoma Cells In Vitro

Glioblastoma multiforme (GBM) is the most lethal subtype of glioma. Cannabis sativa is used for the treatment of various medical conditions. Around 150 phytocannabinoids have been identified in C. sativa, among them Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) that trigger GBM cell death. However, the optimal combinations of cannabis molecules for anti-GBM activity are unknown. Chemical composition was determined using high-performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC/MS). Cytotoxic activity was determined by XTT and lactate dehydrogenase (LDH) assays and apoptosis and cell cycle by fluorescence-activated cell sorting (FACS). F-actin structures were observed by confocal microscopy, gene expression by quantitative PCR, and cell migration and invasion by scratch and transwell assays, respectively. Fractions of a high-THC cannabis strain extract had significant cytotoxic activity against GBM cell lines and glioma stem cells derived from tumor specimens. A standard mix (SM) of the active fractions F4 and F5 induced apoptosis and expression of endoplasmic reticulum (ER)-stress associated-genes. F4 and F5 inhibited cell migration and invasion, altered cell cytoskeletons, and inhibited colony formation in 2 and 3-dimensional models. Combinations of cannabis compounds exert cytotoxic, anti-proliferative, and anti-migratory effects and should be examined for efficacy on GBM in pre-clinical studies and clinical trials

PIF* promotes brain re-myelination locally while regulating systemic inflammation- clinically relevant multiple sclerosis M.smegmatis model.

Neurologic disease diagnosis and treatment is challenging. Multiple Sclerosis (MS) is a demyelinating autoimmune disease with few clinical forms and uncertain etiology. Current studies suggest that it is likely caused by infection(s) triggering a systemic immune response resulting in antigen/non-antigen-related autoimmune response in central nervous system (CNS). New therapeutic approaches are needed. Secreted by viable embryos, PreImplantation Factor (PIF) possesses a local and systemic immunity regulatory role. Synthetic PIF (PIF) duplicates endogenous peptide's protective effect in pre-clinical autoimmune and transplantation models. PIF protects against brain hypoxia-ischemia by directly targeting microglia and neurons. In chronic experimental autoimmune encephalitis (EAE) model PIF reverses paralysis while promoting neural repair. Herein we report that PIF directly promotes brain re-myelination and reverses paralysis in relapsing remitting EAE MS model. PIF crosses the blood-brain barrier targeting microglia. Systemically, PIF decreases pro-inflammatory IL23/IL17 cytokines, while preserving CNS-specific T-cell repertoire. Global brain gene analysis revealed that PIF regulates critical Na+/K+/Ca++ ions, amino acid and glucose transport genes expression. Further, PIF modulates oxidative stress, DNA methylation, cell cycle regulation, and protein ubiquitination while regulating multiple genes. In cultured astrocytes, PIF promotes BDNF-myelin synthesis promoter and SLC2A1 (glucose transport) while reducing deleterious E2F5, and HSP90ab1 (oxidative stress) genes expression. In cultured microglia, PIF increases anti-inflammatory IL10 while reducing pro-inflammatory IFNγ expression. Collectively, PIF promotes brain re-myelination and neuroprotection in relapsing remitting EAE MS model. Coupled with ongoing, Fast-Track FDA approved clinical trial, NCT#02239562 (immune disorder), current data supports PIF's translation for neurodegenerative disorders therapy

Propofol Inhibits Glioma Stem Cell Growth and Migration and Their Interaction with Microglia via BDNF-AS and Extracellular Vesicles

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. GBM contains a small subpopulation of glioma stem cells (GSCs) that are implicated in treatment resistance, tumor infiltration, and recurrence, and are thereby considered important therapeutic targets. Recent clinical studies have suggested that the choice of general anesthetic (GA), particularly propofol, during tumor resection, affects subsequent tumor response to treatments and patient prognosis. In this study, we investigated the molecular mechanisms underlying propofol\u27s anti-tumor effects on GSCs and their interaction with microglia cells. Propofol exerted a dose-dependent inhibitory effect on the self-renewal, expression of mesenchymal markers, and migration of GSCs and sensitized them to both temozolomide (TMZ) and radiation. At higher concentrations, propofol induced a large degree of cell death, as demonstrated using microfluid chip technology. Propofol increased the expression of the lncRNA BDNF-AS, which acts as a tumor suppressor in GBM, and silencing of this lncRNA partially abrogated propofol\u27s effects. Propofol also inhibited the pro-tumorigenic GSC-microglia crosstalk via extracellular vesicles (EVs) and delivery of BDNF-AS. In conclusion, propofol exerted anti-tumor effects on GSCs, sensitized these cells to radiation and TMZ, and inhibited their pro-tumorigenic interactions with microglia via transfer of BDNF-AS by EVs

RTVP-1 regulates glioma cell migration and invasion via interaction with N-WASP and hnRNPK

Glioblastoma (GBM) are characterized by increased invasion into the surrounding normal brain tissue. RTVP-1 is highly expressed in GBM and regulates the migration and invasion of glioma cells. To further study RTVP-1 effects we performed a pull-down assay using His-tagged RTVP-1 followed by mass spectrometry and found that RTVP-1 was associated with the actin polymerization regulator, N-WASP. This association was further validated by co-immunoprecipitation and FRET analysis. We found that RTVP-1 increased cell spreading, migration and invasion and these effects were at least partly mediated by N-WASP. Another protein which was found by the pull-down assay to interact with RTVP-1 is hnRNPK. This protein has been recently reported to associate with and to inhibit the effect of N-WASP on cell spreading. hnRNPK decreased cell migration, spreading and invasion in glioma cells. Using co-immunoprecipitation we validated the interactions of hnRNPK with N-WASP and RTVP-1 in glioma cells. In addition, we found that overexpression of RTVP-1 decreased the association of N-WASP and hnRNPK. In summary, we report that RTVP-1 regulates glioma cell spreading, migration and invasion and that these effects are mediated via interaction with N-WASP and by interfering with the inhibitory effect of hnRNPK on the function of this protein