title: Integrated approaches to miRNAs target definition: time-series analysis in an osteosarcoma differentiative model.
abstract: BACKGROUND
  microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate.
  
  
  METHODS
  Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed.
  
  
  RESULTS
  We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs.
  
  
  CONCLUSIONS
  These data underline that the expression of miR-34a and down-modulation of TGFbeta signaling emerge as pivotal events to drive CD99-mediated reversal of malignancy and activation of differentiation in OS cells. Our results describe crucial and specific interacting actors providing and supporting their relevance as potential targets for therapeutic differentiative strategies.
authors.affiliation: Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, 40136, Bologna, Italy. andrea.grilli@ior.it.
authors.forename: A
authors.initials: A
authors.lastname: Grilli
authors.affiliation: Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, 40136, Bologna, Italy. marika.sciandra@ior.it.|PROMETEO, STB, RIT Department, Rizzoli Orthopedic Institute, Bologna, Italy. marika.sciandra@ior.it.
authors.forename: M
authors.initials: M
authors.lastname: Sciandra
authors.affiliation: Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, 40136, Bologna, Italy. mario.terracciano83@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Terracciano
authors.affiliation: Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, 40136, Bologna, Italy. piero.picci@ior.it.
authors.forename: P
authors.initials: P
authors.lastname: Picci
authors.affiliation: Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, 40136, Bologna, Italy. katia.scotlandi@ior.it.|PROMETEO, STB, RIT Department, Rizzoli Orthopedic Institute, Bologna, Italy. katia.scotlandi@ior.it.
authors.forename: K
authors.initials: K
authors.lastname: Scotlandi
mesh_terms: D000071443:12E7 Antigen; D015703:Antigens, CD / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D002999:Clone Cells / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D010006:Osteoblasts / Q000473:pathology; D012516:Osteosarcoma / Q000235:genetics / Q000473:pathology*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D000071443:12E7 Antigen; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta