Glioblastoma ; genetics ; metabolism ; Humans ; Mutation ; Oncogene Proteins ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism

Patients with glioblastoma (GBM) generally have a bad prognosis and short overall survival after being treated with surgery, chemotherapy or radiotherapy due to the histological heterogeneity, strong invasive ability and rapid postoperative recurrence of GBM. The components of GBM cell-derived exosome (GBM-exo) can regulate the proliferation and migration of GBM cell via cytokines, miRNAs, DNA molecules and proteins, promote the angiogenesis via angiogenic proteins and non-coding RNAs, mediate tumor immune evasion by targeting immune checkpoints with regulatory factors, proteins and drugs, and reduce drug resistance of GBM cells through non-coding RNAs. GBM-exo is expected to be an important target for the personalized treatment of GBM and a marker for diagnosis and prognosis of this kind of disease. This review summarizes the preparation methods, biological characteristics, functions and molecular mechanisms of GBM-exo on cell proliferation, angiogenesis, immune evasion and drug resistance of GBM to facilitate developing new strategies for the diagnosis and treatment of GBM.
Humans ; Glioblastoma/genetics* ; Exosomes/metabolism* ; MicroRNAs/metabolism* ; Prognosis ; Cell Proliferation ; Brain Neoplasms/genetics* ; Cell Line, Tumor

BACKGROUNDSite A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT), consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.

METHODSFifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.

RESULTSThe IDH1 active site included seven residues from chain A (A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp) and three residues from chain B (B214Thr, B212Lys, and B252Asp) that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy (ΔE) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate.

CONCLUSIONSMutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

Catalytic Domain ; genetics ; Glioblastoma ; genetics ; Humans ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Isocitrates ; metabolism ; Mutagenesis ; Mutation ; Protein Binding ; Structure-Activity Relationship

Astrocytoma ; genetics ; metabolism ; Brain Neoplasms ; genetics ; metabolism ; Frizzled Receptors ; genetics ; metabolism ; Glioblastoma ; genetics ; metabolism ; Glioma ; genetics ; metabolism ; Humans ; Paraffin Embedding ; RNA, Messenger ; metabolism ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Signal Transduction ; Wnt2 Protein ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism

Leucine-rich repeats containing 4 ( LRRC4 , also named netrin-G ligand 2 [NGL-2]) is a member of the NetrinGs ligands (NGLs) family. As a gene with relatively high and specific expression in brain, it is a member of the leucine-rich repeat superfamily and has been proven to be a suppressor gene for gliomas, thus being involved in gliomagenesis. LRRC4 is the core of microRNA-dependent multi-phase regulatory loops that inhibit the proliferation and invasion of glioblastoma (GB) cells, including LRRC4/NGL2-activator protein 2 (AP2)-microRNA (miR) 182-LRRC4 and LRRC4-miR185-DNA methyltransferase 1 (DNMT1)-LRRC4/specific protein 1 (SP1)-DNMT1-LRRC4. In this review, we demonstrated LRRC4 as a new member of the partitioning-defective protein (PAR) polarity complex that promotes axon differentiation, mediates the formation and plasticity of synapses, and assists information input to the hippocampus and storage of memory. As an important synapse regulator, aberrant expression of LRRC4 has been detected in autism, spinal injury and GBs. LRRC4 is a candidate susceptibility gene for autism and a neuro-protective factor in spinal nerve damage. In GBs, LRRC4 is a novel inhibitor of autophagy, and an inhibitor of protein-protein interactions involving in temozolomide resistance, tumor immune microenvironment, and formation of circular RNA.
Humans ; Cell Line, Tumor ; Glioblastoma/metabolism* ; Leucine ; Leucine-Rich Repeat Proteins/genetics* ; MicroRNAs ; Nerve Tissue Proteins/genetics* ; Tumor Microenvironment

OBJECTIVETo investigate the effects of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the biological activity of glioblastoma cell line GL15.

METHODSGlioblastoma GL15 cells were cultured and transfected with LRIG3-EGFP plasmid. The location of LRIG3 in GL15 cells was observed with confocal microscopy. The proliferation and invasiveness of GL15 cells were detected with methyl thiazolyl tetrazolium (MTT) and Transwell methods respectively; the expression of epidermal growth factor receptor (EGFR) and LRIG3 mRNA and protein were detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot respectively.

RESULTAfter transfection with the plasmid LRIG-EGFP, LRIG3 fusion protein was found in cytoplasm of GL15 cells and cell proliferative and invasiveness were reduced. The expression of EGFR and LRIG3 varied with the duration of EGF treatment (100 ng/ml): the expression of EGFR decreased while the expression of LRIG3 increased as time prolonged.

CONCLUSIONLRIG3 can inhibit the proliferation and invasiveness of glioblastoma cells and may be used as a target gene in gene therapy of glioblastoma.

Brain Neoplasms ; pathology ; Cell Proliferation ; Epidermal Growth Factor ; genetics ; Glioblastoma ; pathology ; Humans ; Membrane Proteins ; genetics ; metabolism ; Neoplasm Invasiveness ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured

Recent evidence shows that transcriptional silencing as a consequence of hypermethylation of CpG islands is an important mechanism in the inactivation of p16INK4 tumor suppressor gene. This study is designed to clarify the significance of p16INK4 hypermethylation in 23 cases of glioblastomas (GBMs) by methylation-specific polymerase chain reaction (PCR) and p16 immunostaining. Fourteen cases (60.9%) out of 23 GBMs revealed hypermethylation on p16. p16 immunostaining revealed that 13 (93%) of these 14 hypermethylation cases showed complete loss of immunoreactivity and only one (7%) case retained immunoreactivity. Among 9 methylation-negative cases, 4 were immunonegative, which might be related to mutations or deletions other than hypermethylation. The most significant finding was that of 17 cases with immunonegativity, 13 cases (76.5%) showed hypermethylation. We reconfirmed that p16 hypermethylation may be one of the major mechanisms of tumorigenesis of GBMs and the results between the methylation specific-PCR study and p16 immunostaining had a good correlation.
5' Untranslated Regions/metabolism* ; 5' Untranslated Regions/genetics ; Adult ; Antisense Elements (Genetics) ; Brain Neoplasms/pathology ; Brain Neoplasms/genetics* ; Brain Neoplasms/chemistry ; CpG Islands/physiology* ; DNA Methylation* ; Female ; Gene Silencing/physiology ; Glioblastoma/pathology ; Glioblastoma/genetics* ; Glioblastoma/chemistry ; Human ; Male ; Middle Age ; Polymerase Chain Reaction ; Protein p16/genetics* ; Protein p16/analysis

BACKGROUNDEverlasting cellular proliferation is the fundamental feature during gliomagenesis and Ki-67 is one of the classical proliferation markers in human glioblastoma multiforme (GBM). However, the driver genes or core pathways for cellular proliferation in GBM have not been elucidated systematically.

METHODSWe evaluated by immunohistochemistry the prognostic value of Ki-67 expression in the clinical outcome of 156 Chinese patients with GBM and a total of 64 GBM samples were selected for further Agilent genome-wide microarray analysis. On the basis of the microarray data from Tiantan (n = 64) and The Cancer Genome Atlas (TCGA) (n = 202) database, differentially expressed genes between the GBM subgroups with high or low level of Ki-67 expression were identified using Significance Analysis of Microarrays (SAM). Gene Ontology (GO) and KEGG Pathway analyses were then undertaken for the Ki-67 associated genes to identify the most significant biological processes and signaling pathways.

RESULTSWe confirmed that Ki-67 was an independent prognostic indicator in the largest Chinese patient cohort of 156 GBM samples via immunohistochemical staining. Survival analysis of Ki-67 over-expression revealed a highly significant association with a worse clinical outcome (P = 0.010 for progression-free survival; P = 0.007 for overall survival). Comparative and integrated analysis between Tiantan and TCGA database identified a 247-gene "proliferation signature" (205 up-regulated and 42 down-regulated genes) that distinguished Ki-67 expression phenotypes. GO and KEGG Pathway analyses further indicated that Ki-67 expression phenotype was associated with distinct changes in gene expression associated with the regulation of cellular growth and proliferation.

CONCLUSIONSProliferation marker Ki-67 is an independent prognostic indicator in Chinese GBM patients. And Ki-67 associated proliferation signature identified through genome-wide microarray analysis may provide potential targets for anti-proliferation therapy in GBM.

Cell Proliferation ; Computational Biology ; Gene Expression Profiling ; methods ; Glioblastoma ; genetics ; metabolism ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Ki-67 Antigen ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo study the radiation-sensitizing effect of up-regulating p27(kip1) expression by knocking down miR-221/222 in the U251 human glioblastoma cell line.

METHODSBy bioinformatic analysis, we searched the miRNA-221/222 sequence and found the relationship between p27(kip1) and miRNA-221/222. miRNA-221/222 antisense oligonucleotides were transfected into U251 human glioblastoma cells. Northern blot analysis was conducted to detect the expression of miR-221/222 in control, scrambled oligonucleotide (ODN) transfected and anti-mi-221/222ODNs transfected cell groups. The cell cycle kinetics was detected by flow cytometry. Clonogenic assay was used to measure the mitotic cell death and p27(kip1) expression was examined by Western blot analysis.

RESULTSBased on bioinformatic analysis, we found that the seed sequences of miR-221 and miR-222 coincide with each other, and p27(kip1) is a target for miRNA-221/222. The expression level of miR-221/222 was significantly knocked down in cells transfected with antimiR-221/222 as compared to the parental cells or cells transfected with scrambled ODN. Cell cycle was arrested in G0 or G1 phase in the anti-miR-221/222 group. When combined with irradiation, S phase fraction in the anti-miR-221/222 cell group is lower than that in the other two control groups. Anti-miR-221/222 combined with irradiation could synergistically enhance mitotic cell death. The expression of p27(kip1) was up regulated in the anti-miR-221/222 group revealed by Western blot analysis.

CONCLUSIONAnti-miR-221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27(kip1).

Base Sequence ; Cell Cycle ; radiation effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Glioblastoma ; genetics ; metabolism ; Humans ; MicroRNAs ; genetics ; metabolism ; Molecular Sequence Data ; Oligonucleotides, Antisense ; genetics ; metabolism ; Radiation Tolerance ; Sequence Alignment ; Up-Regulation ; radiation effects ; X-Rays

OBJECTIVETo study the effect of silencing Dicer by small interference RNA (siRNA) to suppress the global microRNA (miRNAs) expression on the biological characteristics of TJ905 glioblastoma cells.

METHODSThe silencing effect of RNA interference on Dicer expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining. The cell proliferation rate and cell cycle kinetics were detected by MTT assay and flow cytometry respectively, and the cell invasive ability was evaluated by transwell assay.

RESULTSThe siRNA targeting Dicer suppressed the expression of Dicer in TJ905 cells. Meanwhile, the proliferation activity and invasive ability were significantly enhanced in cells transfected with Dicer siRNA compared to those cells transfected with scrambled siRNA and the control cells.

CONCLUSIONSuppression of Dicer expression renders the glioma cells harboring more aggressive phenotype. This preliminary finding suggests that global lower expression of miRNAs may play an oncogenic role.

Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DEAD-box RNA Helicases ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Glioblastoma ; genetics ; metabolism ; physiopathology ; Humans ; RNA, Small Interfering ; genetics ; metabolism ; Ribonuclease III ; genetics ; metabolism