Medulloblastoma (MB) is the most common malignant mind tumor of pediatric age and is characterized by cells expressing stem astroglial and neuronal markers. Acid- (RA-) induced differentiation. We targeted to identify pivotal players of specific pathways sustaining stemness and/or tumor development and progression and integrate the results of our recent proteomic study. Our results uncovered 22 differentially indicated microRNAs that were used as input together with deregulated genes and proteins in the Genomatix Pathway System (GePS) analysis exposing 3 subnetworks that may be interestingly involved in the maintenance of hMB-SLCs proliferation. Taken together our findings focus on microRNAs genes and proteins that are significantly modulated in hMB-SLCs with respect to their RA-differentiated counterparts and could open fresh perspectives for prognostic and restorative treatment on MB. 1 Intro Aggressive multimodal therapy offers significantly improved medulloblastoma (MB) results but up to 30% of the instances still recur and treated individuals got debilitating secondary sequelae . MB is definitely characterized by significant intratumoral heterogeneity and comprised of cells expressing stem astroglial and neuronal markers whose contribution to tumor development has not been completely understood yet . Our and additional laboratories have offered evidence that MB harbors a distinct subpopulation of stem cells or malignancy stem-like cells (SLCs) [3 4 recognized from the marker manifestation of Nanog . Importantly stem cell signatures have been associated with tumor poor prognosis and very recently we characterized SLCs in MB with aggressive behavior . Interestingly it has been reported that clonal genetic events observed in metastases can be demonstrated inside a restricted subclone of the primary tumor suggesting that only rare cells have the ability Canagliflozin to metastasize . SLCs have been proposed as the major source of resistance toward standard therapy  and a never-ending reservoir for malignancy maintenance and progression . Knowledge of the SLCs molecular features is definitely urgently needed to understand tumor progression and to design novel stem specific therapeutic strategies. About this topic we previously isolated SLCs from human being MBs (hMB-SLCs) [1 3 and more recently investigated the proteomic profile of hMB-SLCs and of their RA-differentiated counterparts applying a label-free quantitative proteomic analysis able to maximize the recognition capacities of the statistically differential spectral features . In MB microRNAs noncoding RNAs that control gene manifestation  have been described as becoming deregulated with respect to normal cerebellum  form regulatory networks with components of signaling pathways deregulated in cancer cells  and have also been described to play a pivotal role in stem Canagliflozin cell differentiation . In new experiments we further characterized expression of microRNA and genes hMB-SLCs Canagliflozin and this paper reports a specific analysis of proteins microRNAs and genes that regulate stem cell maintenance. Since the identification of specific pathways supporting the survival of SLCs could open new perspectives in cancer treatment using the Genomatix Pathway System (GePS) analysis we also performed a deep network pathway Canagliflozin analysis with the aim of building regulatory networks that include the crosstalk among microRNAs mRNAs and proteins to better define SLCs specific signaling components. 2 Materials and Methods 2.1 Materials Unless otherwise indicated media and supplements were purchased from Gibco-Invitrogen (Carlsbad CA) and chemicals were purchased from Sigma-Aldrich (St. Louis MO). 2.2 Culture of hMB-SLCs Human medulloblastoma samples (MB) Pten were collected during surgical resection with the approval of institutional review board as described earlier . Tissues were collected in Hank’s Balanced Salt Solution (HBSS) supplemented with 0.5% glucose and penicillin-streptomycin grossly triturated with serological pipette and treated with DNAse I to a final concentration of 0.04% for 20?min. Subsequently cell aggregates were mechanically disrupted using pipettes of decreasing bore size to obtain a single cell suspension. After dissociation and centrifugation Canagliflozin cells were cultured as oncospheres in selective medium.