Abstract
BackgroundMetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes.ResultsWhole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs’ targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKβ, Birc1, Birc2, Bric5, β-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3β, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1β, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs’ targets corresponded strongly with poor overall and relapse-free survival.ConclusionsFor the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1642-x) contains supplementary material, which is available to authorized users.
Highlights
MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors
Over an extended 50–60 day period, these mice suddenly gained weight and presented with multiple clinically-mimicking metastatic tumors in the mediastinum and retroperitoneal, pelvic, abdominal, and chest cavities (Fig. 1a). These mice generally produced 5–12 large, viable, often multi-lobular tumors in multiple sites with well-organized blood supplies (Fig. 1a). This aggressive disease with metastatic dissemination developed over a short period (1–2 weeks) and vigorously, as Metastatic Site Derived Aggressive cell (MSDAC) clones derived from the manifold of metastatic tumors were discretely characterized by karyotyping, whole genome array CGH analysis, and tumorosphere-forming capacity
Since metastasis is a complex processes, and as we identified a total of 74 metastamiRs in this setting, we examined a total of 49 targets that are involved in tumor progression, i.e., metastasis, in MSDACs and a manifold of metastatic tumors
Summary
MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. We identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. We related their deregulation to the modulation of target proteins and established their association with clinical outcomes. The regulatory role for miRNAs in metastasis has been established [19,20,21,22,23] These miRNAs have been identified as metastamiRs [24], as they have both pro- and anti-metastatic effects. We used a unique, MYCN non-amplified mouse model of human high-risk aggressive metastatic neuroblastoma coupled with whole genome miRNA approach to investigate the functional reorganization of metastamiRs in NB progression
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