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