MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisensemediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.-Evangelisti, C., Florian, M. C., Massimi, I., Dominici, C., Giannini, G., Galardi, S., Buè, M. C., Massalini, S., McDowell, H. P., Messi, E., Gulino, A., Farace, M. G., Ciafrè, S. A. MiR-128 up-regulation inhibits Reelin and DCX expression and reduces neuroblastoma cell motility and invasiveness. FASEB J. 23, 4276 -4287 (2009). www.fasebj.org

Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangements account for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. All mutations were missense and affected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis.

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microRNAs (miRNAs) are a class of regulatory small non-coding molecules that control gene expression at post-transcriptional level. Deregulation of miRNA functions affects a variety of biological processes also involved in the etiology of several human mendelian and complex diseases. Recently, aberrant miRNA expression has been observed in Cystic Fibrosis (CF), an autosomal-recessive genetic disorder caused by mutations in the CFTR gene, in which a genotype-phenotype correlation is not always found. In order to determine miRNA role in CFTR post-transcriptional regulation, we searched for miR-responsive elements in the CFTR 3′-UTR. In silico analysis, performed using different computational on-line programs, identified some putative miRNAs. Both miR-101 and miR-494 synthetic mimics significantly inhibited the expression of a reporter construct containing the 3′-UTR of CFTR in luciferase assays. Interestingly, miR-101/miR-494 combination was able to markedly suppress CFTR activity by approximately 80% (p<0.001). This is one of the first in vitro studies implicating microRNAs as negative regulators of the CFTR gene expression. miRNA aberrant expression and function might explain the wide phenotypic variability observed among CF patients.

Circular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from non-canonical splicing events, which are expressed in all eukaryotes and often conserved among different species. We previously showed that the circRNA originating from the ZNF609 locus (circ-ZNF609) acts as a crucial regulator of human primary myoblast growth: indeed, the downregulation of the circRNA, and not of its linear counterpart, strongly reduced the proliferation rate of in vitro cultured myoblasts. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy. We found that circ-ZNF609 is upregulated in biopsies from the two major RMS subtypes, embryonal (ERMS) and alveolar (ARMS). Moreover, we discovered that in an ERMS-derived cell line circ-ZNF609 knock-down induced a specific block at the G1-S transition, a strong decrease of p-Akt protein level and an alteration of the pRb/Rb ratio. Regarding p-Akt, we were able to show that circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus working as a new regulator of cell proliferation-related pathways. As opposed to ERMS-derived cells, the circRNA depletion had no cell cycle effects in ARMS-derived cells. Since in these cells the p53 gene resulted downregulated, with a concomitant upregulation of its cell cycle-related target genes, we suggest that this could account for the lack of circ-ZNF609 effect in ARMS.

BackgroundRhabdomyosarcoma (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.MethodsUsing 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.ResultsNinety-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.ConclusionsMiR-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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-880) contains supplementary material, which is available to authorized users.

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MicroRNAs (miRNAs) have recently emerged as important gene regulators in Cystic Fibrosis (CF), a common monogenic disease characterized by severe infection and inflammation, especially in the airway compartments. In the current study, we show that both miR-145 and miR-494 are significantly up-regulated in nasal epithelial tissues from CF patients compared with healthy controls (p<0.001 and p<0.01, respectively) by Quantitative Real-Time PCR. Only miR-494 levels showed a trend of correlation with reduced CFTR mRNA expression and positive sweat test values, supporting the negative regulatory role of this miRNA on CFTR synthesis. Using computational prediction algorithms and luciferase reporter assays, SMAD family member 3 (SMAD3), a key element of the TGF-β1 inflammatory pathway, was identified as a target of miR-145. Indeed, miR-145 synthetic mimics suppressed by approximately 40% the expression of a reporter construct containing the SMAD3 3'-UTR. Moreover, we observed an inverse correlation between SMAD3 mRNA expression and miR-145 in CF nasal tissues (r=-0.68, p=0.0018, Pearson's correlation). Taken together, these results confirm the pivotal role of miRNAs in the CF physio-pathogenesis and suggest that miRNA deregulation play a role in the airway disease severity by modulating CFTR levels as well as the expression of important molecules involved in the inflammatory response. miR-494 and miR-145 may, therefore, be potential biomarker and therapeutic target to specific CF clinical manifestations.