Identification of a miRNAs signature as potential biomarker of mesenchymal phenotype in neuroblastoma patients

Neuroblastoma (NB) is a heterogeneous tumor, ranging from cases with spontaneous regression (MS stage) to high risk (HR) tumors. Resistance to therapy presents a major challenge in the treatment and management of HR-NB, contributing to a poor prognosis. In these patients, the resistance to conventional treatment is also related to non-genetic features, such as the cellular plasticity. Novel studies have demonstrated the presence of two distinct cell phenotypes: adrenergic (ADRN) and mesenchymal (MES), which reflect the heterogeneity of NB. MicroRNAs (miRNAs) are small, endogenous and non-coding RNAs with the ability to regulate gene expression and may have a crucial role in controlling cell plasticity. However, the role of miRNAs in NB plasticity has not been investigated yet. We investigated miRNA signature in NB cells subtypes (MES and ADRN) and in the extracellular vesicles (EVs) released by them, to identify potential MES related biomarkers. Differentially expressed miRNAs were identified by RT-qPCR and subjected to gene ontology, KEGG pathway, and protein-protein interaction network analyses. Candidate miRNAs were validated in plasma-derived EVs from NB patients. We identified miR-199a-3p as strongly upregulated in the MES cells subtype. Moreover, its expression levels were significantly higher in primary cell lines derived from HR patients compared to low-risk (LR) ones. This was confirmed by a bioinformatics analysis in patient tissue obtained from TARGET NB dataset. Protein-protein interaction analysis uncovered a complex network, with FN1, CD44, and YAP1, identified as key genes upregulated by miR-199a-3p, all of which are closely associated with the MES phenotype. Among the miRNAs significantly upregulated in EVs derived from MES cell lines, miR-584a-5p was significantly higher in EVs isolated from plasma of HR and L/Intermediate(I)R patients compared to MS. MiR-584-5p is typically considered a tumor suppressor; to support this role miR-584a-3p resulted significantly upregulated in L/IR tumor tissue, both in the INSS and COG classifications (TARGET NB database). Our findings identified specific miRNAs as MES phenotype related biomarkers. Further studies should investigate the potential impact of miRNAs on plasticity-related pathways in order to open new therapeutic strategies.