title: Deep Sequencing the microRNA profile in rhabdomyosarcoma reveals down-regulation of miR-378 family members.
abstract: BACKGROUND
  Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. MicroRNAs (miRNAs) represent a class of short, non-coding, regulatory RNAs which play a critical role in different cellular processes. Altered miRNA levels have been reported in human cancers, including RMS.
  
  
  METHODS
  Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Q-PCR, MTT, cytofluorimetry, migration assay, western blot and immunofluorescence experiments were carried out to determine the role of miR-378a-3p in cancer cell growth, apoptosis, migration and differentiation. Bioinformatics pipelines were used for miRNA target prediction and clustering analysis.
  
  
  RESULTS
  Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS. MiR-378a-3p over-expression in an RMS-derived cell line suppressed IGF1R expression and affected phosphorylated-Akt protein levels. Ectopic expression of miR-378a-3p caused significant changes in apoptosis, cell migration, cytoskeleton organization as well as a modulation of the muscular markers MyoD1, MyoR, desmin and MyHC. In addition, DNA demethylation by 5-aza-2'-deoxycytidine (5-aza-dC) was able to up-regulate miR-378a-3p levels with a concomitant induction of apoptosis, decrease in cell viability and cell cycle arrest in G2-phase. Cells treated with 5-aza-dC clearly changed their morphology and expressed moderate levels of MyHC.
  
  
  CONCLUSIONS
  MiR-378a-3p may function as a tumour suppressor in RMS and the restoration of its expression would be of therapeutic benefit in RMS. Furthermore, the role of epigenetic modifications in RMS deserves further investigations.
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy. francesca.megiorni@uniroma1.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Megiorni
authors.forename: Samantha
authors.initials: S
authors.lastname: Cialfi
authors.forename: Heather P
authors.initials: HP
authors.lastname: McDowell
authors.forename: Armando
authors.initials: A
authors.lastname: Felsani
authors.forename: Simona
authors.initials: S
authors.lastname: Camero
authors.forename: Alessandro
authors.initials: A
authors.lastname: Guffanti
authors.forename: Barry
authors.initials: B
authors.lastname: Pizer
authors.forename: Anna
authors.initials: A
authors.lastname: Clerico
authors.forename: Alessandra
authors.initials: A
authors.lastname: De Grazia
authors.forename: Antonio
authors.initials: A
authors.lastname: Pizzuti
authors.forename: Anna
authors.initials: A
authors.lastname: Moles
authors.forename: Carlo
authors.initials: C
authors.lastname: Dominici
mesh_terms: D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D019175:DNA Methylation; D015536:Down-Regulation; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D024510:Muscle Development; D017526:Receptor, IGF Type 1; D017451:Receptors, Somatomedin / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA
chemical_list: C000597236:IGF1R protein, human; C526012:MIRN378 microRNA, human; D035683:MicroRNAs; D017451:Receptors, Somatomedin; D017526:Receptor, IGF Type 1