OBJECTIVES: To investigate the mechanism by which circ_EPHB4 regulates temozolomide (TMZ) sensitivity of glioma cells through the miR-424-5p/Wnt3 signal axis. METHODS: We detected the expression levels of circ_EPHB4, miR-424-5p and Wnt3 mRNA in glioma specimens from 25 patients with primary glioma and 25 patients experiencing relapse following temozolomide-based chemotherapy and in TMZ-sensitive and -resistant glioma A172 and SHG44 cells with circ_EPHB4 knockdown using qRT-PCR, and Wnt3 protein expression level was detected with Western blotting. Cell viability, colony-forming ability, and apoptosis of the cells with circ_EPHB4 knockdown were assessed, and the targeted regulation relationship between circ_EPHB4, miR-424-5p, and Wnt3 was verified by dual luciferase reporter assay and RNA immunoprecipitation (RIP) experiments. The effect of circ_EPHB4 knockdown on tumorigenesis of glioma cells was evaluated in subcutaneous tumor-bearing nude mouse models. RESULTS: The expression of circ_EPHB4 was significantly increased in glioma tissues and cells as compared with normal neural tissues and astrocytes (P=0.014). In TMZ-resistant glioma cells, circ_EPHB4 knockdown resulted in an obvious reduction of IC50 value of TMZ, inhibited cell colony formation, and promoted cell apoptosis, and these effects were reversed by miR-424-5p knockdown. The expressions of miR-424-5p and circ_EPHB4 were negatively correlated in glioma tissues (P=0.011). MiR-424-5p knockdown also attenuated the effect of circ_EPHB4 knockdown on expressions of PCNA, P-gp, MRP1 and bax. CONCLUSIONS Circ_EPHB4 regulates Wnt3 expression through "sponge adsorption" of miR-424-5p, thereby modulating TMZ-resistant glioblastoma cell clonogenesis, apoptosis, and TMZ sensitivity, suggesting the potential of circ_EPHB4 as a therapeutic target for reversing drug resistance of gliomas.
MicroRNAs/genetics* ; Humans ; Temozolomide ; Glioma/genetics* ; Animals ; Mice, Nude ; Cell Line, Tumor ; Wnt3 Protein/metabolism* ; Mice ; Apoptosis ; RNA, Circular ; Drug Resistance, Neoplasm ; Brain Neoplasms/pathology* ; Signal Transduction

OBJECTIVES: To investigate the oncogenic role of circular RNA circ_EPHB4 in glioma and its molecular mechanism. METHODS: Microarray analysis was performed to identify the differentially expressed circRNAs in glioma tissues. The effects of circ_EPHB4 on glioma cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and tumorigenicity in vivo were assessed using scratch wound healing assay, Transwell invasion assay and nude mouse models bearing subcutaneous tumors. RNA immunoprecipitation (RIP), RNA stability assays, and gene overexpression and silencing techniques were employed to validate the synergistic regulatory effect of circ_EPHB4 and the N6-methyladenosine (m⁶A) reader protein YTHDF3 on Wnt3 expression. RESULTS: Circ_EPHB4 was significantly overexpressed by 2.3 folds (|log2FC|=1.2, P<0.01) in glioma tissues compared to the adjacent tissues, and by 2.5 folds in glioma cell line U373 compared to normal cells (P<0.001). Overexpression of circ_EPHB4 significantly enhanced migration and invasion of glioma cells, and promoted the expressions of EMT markers N-cadherin and vimentin. In the tumor-bearing mouse models, the tumor volume in circ_EPHB4 overexpression group was significantly greater than that in the control group, and the lung metastatic foci increased by 4.2 folds. Overexpression of circ_EPHB4 promoted oncogenesis by upregulating Wnt3 expression, while YTHDF3 extended the half-life of Wnt3 mRNA in an m⁶A-dependent manner. Simultaneous knockdown of circ_EPHB4 and YTHDF3 resulted in an obvious reduction of Wnt3 mRNA expression by up to 47% compared to its level following knocking down either circ_EPHB4 or YTHDF3 alone. CONCLUSIONS Circ_EPHB4 and YTHDF3 promote glioma progression by jointly targeting the Wnt3 signaling pathway, which may provide a new therapeutic strategy for gliomas.
Glioma/genetics* ; Humans ; Animals ; Cell Line, Tumor ; RNA-Binding Proteins/genetics* ; RNA, Circular ; Epithelial-Mesenchymal Transition ; Mice, Nude ; Cell Movement ; Wnt3 Protein/genetics* ; Mice ; Disease Progression ; Adenosine/metabolism* ; Brain Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic

Neuroplastin 65 (Np65) is an immunoglobulin superfamily cell adhesion molecule involved in synaptic formation and plasticity. Our recent study showed that Np65-knockout (KO) mice exhibit abnormal cognition and emotional disorders. However, the underlying mechanisms remain unclear. In this study, we found 588 differentially-expressed genes in Np65-KO mice by microarray analysis. RT-PCR analysis also revealed the altered expression of genes associated with development and synaptic structure, such as Cdh1, Htr3a, and Kcnj9. In addition, the expression of Wnt-3, a Wnt protein involved in development, was decreased in Np65-KO mice as evidenced by western blotting. Surprisingly, MRI and DAPI staining showed a significant reduction in the lateral ventricular volume of Np65-KO mice. Together, these findings suggest that ablation of Np65 influences gene expression, which may contribute to abnormal brain development. These results provide clues to the mechanisms underlying the altered brain functions of Np65-deficient mice.
Affective Symptoms ; metabolism ; Animals ; Brain ; diagnostic imaging ; metabolism ; pathology ; Cognition Disorders ; metabolism ; Gene Expression ; Magnetic Resonance Imaging ; Membrane Glycoproteins ; deficiency ; genetics ; physiology ; Mice, Knockout ; Microarray Analysis ; Organ Size ; Real-Time Polymerase Chain Reaction ; Wnt3 Protein ; metabolism

OBJECTIVETo investigate the effects of adenoviral vector-mediated over-expression of Wnt3 on the apoptosis of hepatic progenitor cells in vitro.

METHODSHepatic progenitor cells transfected with Ad-GFP-Wnt3 vector or the control vector Ad-GFP were examined for cell apoptosis under fluorescence microscopy with Hoechst 33342 staining, and the proportion of apoptotic cells were determined by flow cytometric analysis with Annexin-PE/7-ADD staining. The mRNA and protein expressions of Bax, Bcl-2 and Bcl-xl in the cells were detected by real-time PCR and Western blotting, respectively.

RESULTSReal-time PCR and Western blotting showed a high expression of Wnt3 in Ad-GFP-Wnt3-transfected hepatic progenitor cells, which exhibited significantly decreased cell apoptosis as compared with the control group. The expressions of Bcl-2 and Bcl-xl mRNA and proteins increased significantly while Bax expression decreased obviously in Ad-GFP-Wnt3-transfected cells (P<0.05).

CONCLUSIONSAdenoviral vector-mediated over-expression of Wnt3 can suppress apoptosis of hepatic progenitor cells possibly through the Bcl-2 pathway.

Apoptosis ; Cells, Cultured ; Genetic Vectors ; Hepatocytes ; cytology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stem Cells ; cytology ; Transfection ; Wnt3 Protein ; metabolism ; bcl-X Protein ; metabolism

As a member of the hox gene family, hoxB4 gene encodes a class of DNA-dependent homeobox domain nucleoprotein, which is a specific transcription factor, playing an important role in regulating the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Therefore, it is important to understand the mechanisms involved in regulating expression of hoxB4 in the HSC. Previous studies have suggested that some hoxB4 upstream regulatory factors, such as USF-1 (upstream activating factor -1), USF-2 (upstream activating factor -2) and NF-Y complex, as well as hematopoietic cytokines, such as platelet growth factor (TPO) and Wnt3a protein, play important regulatory roles in the expression of hoxB4 in hematopoietic stem cells. In this review the structure and biological characteristics of hoxB4, mechanisms involved in regulating expression of hoxB4 in the HSC are summarized.
CCAAT-Binding Factor ; metabolism ; Gene Expression Regulation ; Genes, Homeobox ; genetics ; physiology ; Hematopoietic Stem Cells ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; Humans ; Transcription Factors ; genetics ; metabolism ; physiology ; Upstream Stimulatory Factors ; metabolism ; Wnt Proteins ; metabolism ; Wnt3 Protein ; Wnt3A Protein

OBJECTIVEPrevious studies suggest that hyperbaric oxygen (HBO) treatment promotes the proliferation of neurocytes in neonatal rats following hypoxic-ischemic brain damage (HIBD). The Wnt signaling pathway is associated with neurogenesis. This study examined whether HBO promoted neural stem cells (NSCs) proliferation after HIBD, and whether that the proliferation correlated with Wnt-3 protein expression.

METHODSSeven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control, hypoxia-ischemia (HI), and HI-HBO. HI was induced by the ligation of left common carotid artery, followed by a 2-hr exposure to 8% O2 in the latter two groups. HBO was administered 3 hrs after HI in the HI-HBO group for continuous 7 days (2 atmospheres absolute, once daily). The proliferating NSCs in the subventricular zone (SVZ) was examined by BrdU/nestin immunofluorescence and the expression of Wnt-3 protein in NSCs was examined by nestin/Wnt-3 immunofluorescence at 6 and 24 hrs and at 3, 7 and 14 days of HI. The cellular expressions of nestin and Wnt-3 protein were analyzed by laser scanning confocal microscopy. The linear regression analysis was used to evaluate the correlation between cellular Wnt-3 and nestin protein. The expressions of nestin and Wnt-3 protein in the ischemic cerebral hemisphere were analyzed with Western blotting.

RESULTSThe number of BrdU/nestin positive cells in the SVZ increased 3 hrs after HBO therapy, peaked at 7 days and remained at a higher level until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of Wnt-3 protein in NSCs increased significantly 3 hrs after HBO therapy, peaked at 3 days and remained at high levels until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of cellular nestin protein was closely correlated with the level of cellular Wnt-3 protein (r = 0.893, P < 0.05). The Western blotting analysis demonstrated increased Wnt-3 and nestin protein expressions in the ischemic cerebral hemispheres.

CONCLUSIONSHBO treatment promotes the proliferation of NSCs in HIBD neonatal rats, which is correlated with the activation of Wnt signaling.

Animals ; Animals, Newborn ; Blotting, Western ; Bromodeoxyuridine ; metabolism ; Cell Proliferation ; Female ; Fluorescent Antibody Technique ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Intermediate Filament Proteins ; Male ; Nerve Tissue Proteins ; Nestin ; Neurons ; cytology ; Rats ; Stem Cells ; cytology ; Wnt Proteins ; analysis ; Wnt3 Protein