download report - Istituto Pasteur

P a r t i c i p a n t s :
Anna Guarini, Franca Citarella, researchers; Sabina
Chiaretti, post-doc fellow; Marilisa Marinelli, Monica
Messina, Nadia Peragine, Simona Santangelo, PhD students.
Report of activity
Background
MicroRNAs (miRNAs) are endogenous, non-coding
small RNAs that negatively regulate gene expression
in a sequence specific manner via translational
repression and/or mRNA degradation. The elevated
tissue-specific expression of some miRNA genes
suggests that they might be involved in tissue differentiation
and maintenance of cell-type identity in
animals; miRNAs would share such a role with tissue-specific
transcriptional factors. Several studies
have reported distinct patterns of miRNA expression
in different hematopoietic cell lineages.
Aims
The aim of the study was the evaluation of the role
of miRNAs as modulators of the signal transduction
in neoplastic B cells, obtained from chronic lymphocytic
leukemia (CLL) patients compared to normal B
cells upon BCR stimulation and the correlation of
these results with the gene profile expression of
IgM stimulated neoplastic and normal B cells. The
study is built on the hypothesis that BCR signaling
plays an important role in the proliferation and
maintenance of the malignant B-cell clone in
patients with CLL. This implies that the key to the
distinctive behavior of the neoplastic cell most likely
relates with the differential ability of the BCR to
respond to stimuli. It is, therefore, of primary relevance
to investigate how the interactions of the BCR
with environmental stimuli may contribute to the
differential disease progression and prognosis that
lead to an heterogeneous clinical course characteris-
97
Cellular and molecular immunology - AREA 5
Potential role of miRNAS in IgM-mediated signal transduction in
normal and neoplastic B cells
Principal investigator: Roberto Foà
Professor of Hematology
Dipartimento di Biotecnologie Cellulari ed Ematologia
Sezione di Ematologia
Tel: (+39) 06 85795753; Fax: (+39) 06 85795792
rfoa@bce.uniroma1.it
tic of CLL patients. All these events could be regulated
by miRNAs that play a significant role in the
proliferation and differentiation of hematopoietic
cells, and it is well-known that changes in miRNA
expression may contribute to cancer predisposition
and progression in cells of the immune system.
Patients and methods
Eighteen patients with a diagnosis of CLL based on
the presence of more than 5,000 peripheral lymphocytes/µL
expressing a typical phenotype (CD5/
CD20/CD19/CD23+, weak surface immuno-globulin
expression, CD10-), have so far been evaluated
before any treatment. The implication that some biological
predictor factors have a role with the progression
of the disease suggested the investigation
of these markers. Thus, CLL cells were evaluated for
the expression of prognostic parameters: CD38 and
ZAP-70 expression; deletion of the 13q14, 11q23,
17p13 regions and chromosome 12 trysomy; the
mutational status of the immunoglobulin variable
genes (IgVH ).
Twelve healthy donors have been utilized as controls.
Normal and leukemic cells have been selected from
peripheral blood using CD19+ microbeads and the
obtained purity was greater than 98%. The purified
cells were cultured in 96 well U bottom plates coated
overnight at 4°C with 50µg/ml anti-goat F(ab’)2
IgG developed in rabbit. BCR stimulation was performed
by adding a goat F(ab’)2 anti-human IgM at
a final concentrations of 10µg/ml for 24 and 48
hours; stimulated and unstimulated cells were thereafter
collected. Some cells were lysed and total RNA
was extracted for gene profile analysis, for miRNA
detection and for validation RQ-PCR tests. miRNA
expression profiling was performed using a service
provider (LC Sciences), while gene expression profiling
analysis was performed using the Affymetrix
HGU133 Plus 2.0 gene chips arrays. Stimulated and
unstimulated CLL cells were evaluated also for apoptosis
and cell cycle.