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I. Bozzoni - RNA-RNA and RNA-protein interactions in the cell nucleus<br />
of NFI-A. In turn, the decrease in NFI-A levels is<br />
important for the activation of differentiation-specific<br />
genes such as M-CSFr. In line with these data,<br />
both RNAi against NFI-A and ectopic expression of<br />
miR-424 precursor cells enhance monocytic differentiation.<br />
The interplay among these three components<br />
was found in APL cell lines as well as in culture of<br />
human CD34 + progenitors differentiating selectively<br />
through the monocyte/macrophage lineage. These<br />
data point to the important role of miRNAs in conjunction<br />
to master transcription factors in<br />
hematopoietic differentiation and indicate the crucial<br />
role of NFI-A in counteracting the monocyte differentiation<br />
programme (Rosa et al., 2007).<br />
Role of miRNAs in neuronal tumorigenesis<br />
mir-9, mir-125a and mir-125b have been shown to<br />
be up-regulated upon in vitro differentiation of<br />
neuroblastoma cell lines (Laneve et al., Proc Natl<br />
Acad Sci USA 2007, 104:7957-62). By two complementary<br />
approaches, ectopic expression and knockdown<br />
experiments, the role of these miRNAs in<br />
molecular circuitries contributing to neuronal cancerogenesis<br />
was clarified. We showed that the overexpression<br />
of the three miRNAs caused a strong<br />
reduction of cell growth; accordingly, microRNAs<br />
were down-regulated in human primary neuroblastomas,<br />
suggesting a tumor suppressor activity for<br />
these molecules.<br />
By bioinformatics and experimental data, we dissected<br />
the molecular mechanisms involving such<br />
miRNAs, and found a new regulatory circuitry controlling<br />
neuroblastoma cell growth. We have identified<br />
the truncated, enzimatically inactive, isoform of<br />
the neurotrophin-3 receptor trkC as their target<br />
gene and demonstrated that miRNA-mediated downregulation<br />
of this protein was crucial for controlling<br />
neuroblastoma cell growth.<br />
Furthermore, the first miRNA expression profiling<br />
of human primary medulloblastomas was performed<br />
by high throughput analysis; we found 78 miRNAs<br />
displaying differential expression between tumors<br />
34<br />
and controls, indicating that specific miRNA signatures<br />
may distinguish tumors from normal tissues.<br />
Use of sncRNA for the gene therapy of<br />
Duchenne Muscular Dystrophy<br />
The use of the mdx mouse as the animal model for<br />
the Duchenne Muscular Dystrophy has allowed us to<br />
demonstrate the effectiveness of the antisense strategy<br />
in restoring the synthesis of dystrophin in vivo.<br />
In the last year we have been able to show that persistent<br />
exon-skipping in mdx mice is maintained for<br />
the entire life of the animal through the use of<br />
Adeno-associated viral (AAV). The transduced muscles<br />
rescued dystrophin expression and displayed a<br />
significant recovery of function towards the normal<br />
values at single muscle fibre level. Therefore, this<br />
approach provides solid bases for a systemic use of<br />
AAV-mediated antisense-U1 snRNA expression for<br />
the therapeutic treatment of DMD.<br />
Antisense constructs specific for human mutations<br />
are currently under construction and testing in<br />
myoblasts from patient biopsies.<br />
Selected publications<br />
Rosa A, Ballarino M, Sorrentino A, Sthandier O, De<br />
Angelis FG, Marchioni M, Guarini A, Fatica A,<br />
Peschle C, Bozzoni I. The interplay between the master<br />
transcription factor PU.1 and miR-424 regulates<br />
human monocyte/macrophage differentiation. Proc<br />
Natl Acad Sci USA 2007, 104:19849-54.<br />
Denti MA, Incitti T, Sthandier O, Nicoletti C, De<br />
Angelis FG, Rizzato E, Auricchio A, Musarò A,<br />
Bozzoni I. Long-term benefit of adeno-associated<br />
virus/antisense-mediated exon skipping in dystrophic<br />
mice. Hum Gene Ther. 2008, 19:601-8.<br />
Morlando M, Ballarino M, Gromak N, Pagano F,<br />
Bozzoni I, Proudfoot NJ. Primary microRNA transcripts<br />
are processed co-transcriptionally. Nat Struct<br />
Mol Biol. 2008, 15:902-9.