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

Amanda Tivnan 0 2 Lorraine Tracey 1 Patrick G Buckley 0 2 Leah C Alcock 0 2 Andrew M Davidoff 1 3 Raymond L Stallings 0 2 0 Department of Cancer Genetics, Royal College of Surgeons in Ireland , York House, York Street, Dublin 2 , Ireland 1 Department of Surgery, St. Jude Children's Research Hospital , Memphis, TN 38105 , USA 2 National Children's Research Centre, Our Lady's Children's Hospital , Crumlin, Dublin 12 , Ireland 3 Department of Surgery, University of Tennessee Health Science Center , Memphis, TN 38105 , USA 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-NASluc 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 2 tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm ). 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. - Background MicroRNAs (miRNAs) are noncoding RNA molecules which act as post-transcriptional regulators of specific messenger RNA transcripts (mRNAs), resulting in targeted degradation and suppression of gene expression. MiRNAs play major roles in normal developmental processes [1,2], and their dysregulation significantly contributes to various aspects of carcinogenesis in nearly all forms of cancer, negatively regulating both tumor suppressor and oncogenes [3]. As reviewed by Stallings et al., miRNAs play particularly important roles in the pathogenesis of neuroblastoma, a paediatric cancer originating from precursor cells of the sympathetic nervous system [4,5]. Neuroblastomas are particularly problematic in that some genetic subtypes, such as those exhibiting amplification of the MYCN oncogene or deletion of chromosome 11q, are associated with very poor patient survival in spite of intensive multimodal chemotherapy. MiR-34a maps to the distal region of chromosome 1p which is commonly deleted in neuroblastoma and was first identified as having a tumor suppressive function in neuroblastoma [6]. Tumors with loss of 1p are more commonly of the MYCN amplified variety (see Stallings for review [7]). Specifically, in the study by Welch et al., [6] and in later studies [8,9], ectopic over-expression of miR-34a in neuroblastoma cell lines resulted in the activation of a caspase-mediated apoptotic pathway. The importance of miR-34a in cancer is now firmly established, having tumor suppressive effects in multiple types of cancer, including leukemias [10], hepatocellular carcinoma [11], pancreatic [12] and colon [13], among others. MiR-34a has multiple experimentally validated targets involved with cellular proliferation and apoptosis, such as MYCN, BCL2, SIRT1, SFRP1 CAMTA1, NOTCH1, JAG1, CCND1, CDK6 and E2F3 [6,9,14-19]. Notably, miR-34a is directly up-regulated by p53 [14,20-22] and a related family member, miR-34c, also has tumor suppressive affects [8,23,24]. Although the dir (...truncated)