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    MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma

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    Authors
    Tivnan, Amanda
    Tracey, Lorraine
    Buckley, Patrick G
    Alcock, Leah C
    Davidoff, Andrew M
    Stallings, Raymond L
    Issue Date
    2011-01-25
    
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    Citation
    BMC Cancer. 2011 Jan 25;11(1):33
    URI
    http://hdl.handle.net/10147/122151
    Abstract
    ABSTRACT Background Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis. Methods A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm2). Results Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved. Most notably, in vivo studies showed that tumor growth was significantly repressed after exogenous miR-34a administration in retroperitoneal neuroblastoma tumors. Conclusion We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.
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