MiR-18a regulates the proliferation,migration and invasion of human glioblastoma cell by targeting neogenin

MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the association of miR-18a, an important member of this cluster, with glioblastoma remains unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human astrocytes, and the expression levels were increased along with the rising pathological grades of glioblastoma. Neogenin was identified as the target gene of miR-18a by dual-luciferase reporter assays. RT-PCR and western blot results showed that its expression levels were decreased along with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly up-regulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma.     

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

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Neogenin Interacts with Matriptase-2 to Facilitate Hemojuvelin Cleavage

Hemojuvelin (HJV) and matriptase-2 (MT2) are co-expressed in hepatocytes, and both are essential for systemic iron homeostasis. HJV is a glycosylphosphatidylinositol-linked membrane protein that acts as a co-receptor for bone morphogenetic proteins to induce hepcidin expression. MT2 regulates the levels of membrane-bound HJV in hepatocytes by binding to and cleaving HJV into an inactive soluble form that is released from cells. HJV also interacts with neogenin, a ubiquitously expressed transmembrane protein with multiple functions. In this study, we showed that neogenin interacted with MT2 as well as with HJV and facilitated the cleavage of HJV by MT2. In contrast, neogenin was not cleaved by MT2, indicating some degree of specificity by MT2. Down-regulation of neogenin with siRNA increased the amount of MT2 and HJV on the plasma membrane, suggesting a lack of neogenin involvement in their trafficking to the cell surface. The increase in MT2 and HJV upon neogenin knockdown was likely due to the inhibition of cell surface MT2 and HJV internalization. Analysis of the Asn-linked oligosaccharides showed that MT2 cleavage of cell surface HJV was coupled to a transition from high mannose oligosaccharides to complex oligosaccharides on HJV. These results suggest that neogenin forms a ternary complex with both MT2 and HJV at the plasma membrane. The complex facilitates HJV cleavage by MT2, and release of the cleaved HJV from the cell occurs after a retrograde trafficking through the TGN/Golgi compartments.     

The Netrin receptor Neogenin is required for neural tube formation and somitogenesis in zebrafish

The Netrin receptor Deleted in colon cancer (Dcc) has been shown to play a pivotal role in the guidance of nascent axons towards the ventral midline in the developing nervous systems of both vertebrates and invertebrates. In contrast, the function during embryogenesis of a second Dcc-like Netrin receptor Neogenin has not yet been defined. We used antisense morpholino oligonucleotides to knockdown Neogenin activity in zebrafish embryos and demonstrate that Neogenin plays an important role in neural tube formation and somitogenesis. In Neogenin knockdown embryos, cavitation within the neural rod failed to occur, producing a neural tube lacking a lumen. Somite formation was also defective, implicating Neogenin in the migration events underlying convergent extension during gastrulation. These observations suggest a role for Neogenin in determining cell polarity or migrational directionality of both neuroectodermal and mesodermal cells during early embryonic development.     

Overexpression of repulsive guidance molecule (RGM) a induces cell death through Neogenin in early vertebrate development

Repulsive guidance molecule (RGM) a is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein that has been implicated in chemorepulsive axon guidance. Although RGMa binds the transmembrane receptor Neogenin, the developmental events controlled by the RGMa-Neogenin interactions in vivo remain largely unknown. We have cloned full-length RGMa from Xenopus borealis for the first time and identified two homologous genes referred to as RGMa1 and RGMa2. Here we show RGMa1 overexpression at 2-cell-stage resulted in cell death, which lead to an early embryonic lethal phenotype of the embryos. Time-lapse photomicroscopy revealed that embryos began to show initial morphological defects from ∼5 h post-fertilization (hpf) which was then followed by extensive blastomere cell death at ∼11 hpf. This phenotype was rescued by simultaneous knock down of RGMa using translation blocking anti-sense morpholinos. Knock down of the RGMa1 receptor Neogenin in RGMa1 overexpressing embryos was also able to rescue the phenotype. Together these results indicated that RGMa1 was signalling through Neogenin to induce cell death in the early embryo. While previous studies have suggested that Neogenin is a dependence receptor that induces cell death in the absence of RGM, we have instead shown that Neogenin-RGM interactions induce cell death in the early embryo. The roles of RGMa1 and Neogenin appear to be context specific so that their co-ordinated and regulated expressions are essential for normal development of the vertebrate embryo.