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L’analisi integrata dei profili di espressione miRNA-mRNA rivela l’alterazione di network funzionali in cellule CD34+ di mielofibrosi primaria
No full textTra le neoplasie mieloproliferative Philadelphia negative, la mielofibrosi primaria (PMF) è il disordine più aggressivo. E’ caratterizzata da fibrosi del midollo osseo (BM), emopoiesi extramidollare, mobilizzazione dei progenitori emopoietici e mieloproliferazione, a carico sia del lineage granulocitario che megacariocitario (MK). Nonostante la PMF sia un’entità clinica nota dal 1951, solamente nel 2005 sono state gettate le basi per la comprensione dei meccanismi molecolari alla base di questa patologia, con la scoperta della mutazione JAK2V617F, presente nel 60-70% dei pazienti affetti da PMF. Da allora, sono state scoperte altre mutazioni, come sui geni MPL, CALR, ASXL1, DNMT3A. Nonostante ciò, manca ancora un quadro definito dei meccanismi patogenetici alla base della PMF. Recentemente, sono stati identificati ulteriori meccanismi molecolari: l’espressione aberrante di microRNAs (miRNAs) sembra avere un ruolo importante poiché sono state riportate signatures di miRNA che discriminano cellule di PMF dalle normali. Per studiare la deregolazione dei miRNA nella PMF, ne abbiamo ottenuto il profilo di espressione da cellule CD34+ provenienti da 31 donatori sani (16 da sangue periferico e 15 da BM) e 42 pazienti affetti da PMF, utilizzando l’array Affimetrix miRNA 2.0. Tra i miRNA modulati, ve ne sono diversi già descritti nell’emopoiesi normale o maligna; in particolare, tra i miRNA up-regolati più interessanti, vi è miR-21-5p è up-regolato nelle cellule CD34+ di PMF e il modello murino di overespressione midollare sviluppa linfomi. Dal momento che i miRNAs agiscono indirettamente nei processi biologici bersagliando mRNAs, abbiamo eseguito un’analisi integrata (IA) con il profilo di espressione genica, ottenuto nel nostro laboratorio dalla stessa popolazione cellulare. L’IA è stata eseguita con il software Ingenuity Pathway Analysis, in grado di combinare i target predetti in silico con i dati di espressione. Abbiamo così identificato circuiti coinvolti nella disregolazione della trascrizione e del rimodellamento della cromatina, processi correlati all’insorgenza e progressione della PMF. In particolare, abbiamo identificato un network che riunisce diversi oncomiRs (miR-155-5p, miR-19a-3p, miR-29a-3p) che bersagliano geni la cui down-regolazione è stata associata all’emopoiesi maligna (JARID2, NR4A3, CDC42, HMGB3). I dati ottenuti dai saggi di luciferasi 3’UTR hanno confermato un alto potere predittivo dell’IA, provato dalle 11/18 predizioni corrette presenti nel network in esame. Inoltre, poichè la down-regolazione di JARID2 era stata precedentemente associata ad un aumentato differenziamento MK, abbiamo qui dimostrato che l'upregolazione di miR-155-5p promuove un'espansione della linea MK, targettando JARID2. Inoltre, il miRNA up-regolato miR-494 ha suscitato il nostro interesse, poiché bersaglia svariati mRNA down-regolati, la cui diminuita espressione gioca un ruolo importante in varie neoplasie mieloidi. Al fine di studiare il ruolo di miR-494 nella patogenesi della PMF, abbiamo over-espresso questo miRNA in cellule CD34+ normali. La valutazione del ciclo cellulare ha evidenziato un aumento di cellule in fase G0/G1, mentre lo studio del differenziamento ha evidenziato che l’up-regolazione di miR-494 è in grado di forzare il commitment verso la linea MK, coerentemente con il tratto distintivo della PMF di una abnorme proliferazione di questa popolazione. Nel complesso, l’integrazione dei profili di espressione di geni e miRNA ha permesso di identificare network nei quali interagiscono miRNA e geni deregolati, chiarendo alcune caratteristiche patogenetiche della PMF. Infine, l’IA si è dimostrata un approccio biologicamente rilevante per identificare miRNA espressi in modo differenziale, come miR-494, la cui overespressione è potenzialmente coinvolta nella megacariocitopoiesi aberrante.Primary myelofibrosis (PMF) is the most aggressive disorder among Philadelphia-negative myeloproliferative neoplasms. It is characterized by bone marrow (BM) fibrosis, extramedullary haematopoiesis, and myeloproliferation, involving both megakaryocitic (MK) and granulocytic lineages. Despite PMF is well-known clinical entity initially described in 1951, the first insight into the molecular mechanisms underlying this neoplasm was gained only in 2005 with the discovery of somatic gain-of-function mutation JAK2V617F, shared by 60% of PMF patients. Since then, many other mutations were found, such as in MPL, CALR, ASXL1, DNMT3A genes. Nonetheless, a complete framework of the leading pathogenetic mechanisms is still far from being defined. Recently, new molecular mechanisms were uncovered. Among them, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of PMF, as specific miRNA signatures capable of discriminating PMF cells from those of normal donors were previously reported. To have a comprehensive picture of miRNA deregulation in PMF, we profiled miRNA expression in CD34+ cell samples from 31 healthy donors (16 from peripheral blood and 15 from BM) and 42 PMF patients, by means of Affymetrix arrays. Among the miRNAs modulated in PMF, several miRNAs were already described as implicated in normal or malignant hematopoiesis; in particular, among upregulated miRNAs, we found miR-29a-3p, whose overexpression is related to the onset of leukaemia in mice; similarly, miR-21-5p is upregulated in PMF CD34+ cells and a miR-21-5p-overexpressing mouse model develops lymphomas. Moreover, since miRNAs act indirectly in biological processes by targeting mRNAs, we performed an integrative analysis (IA) with the gene expression profile obtained in our lab from the same CD34+ cell population. IA was performed in silico by means of Ingenuity Pathway analysis software, which is able to combine computational predicted targets with gene expression data. Thus, we identified different networks involved in the dysregulation of transcriptional control and chromatin remodeling, potentially related to PMF onset and progression. In particular, we uncovered a circuit gathering several oncomiRs (miR-155-5p, miR-19a-3p, miR-29a-3p) targeting genes whose downregulation has been associated to malignant hematopoiesis (JARID2, NR4A3, CDC42, HMGB3). The data obtained from 3'UTR luciferase assays pointed out a high predictive power of IA, as proved by 11/18 successful predictions for the above-mentioned network. Since JARID2 down-regulation was previously associated with an increased MK commitment, we further demonstrated that upregulated miR-155-5p targets JARID2 in CD34+ cells, thus promoting an expansion of the MK lineage cells. Moreover, upregulated miR-494-3p drew our attention, since it targets the highest number of anti-correlated mRNAs, downregulated in PMF CD34+ cells. In order to investigate the role of miR-494-3p in PMF pathogenesis, we decided to overexpress this miRNA in normal CD34+ cells. Assessment of cell cycle variations highlighted an increase in G0/G1 phase cells, whereas evaluation of cell differentiation unveiled that miR-494-3p enforced expression was able to skew the haematopoietic commitment towards the MK lineage, consistently with the distinctive feature of PMF of an abnormally increased MK population. As a whole, integration of gene and miRNA expression profiles uncovered regulatory networks in which aberrantly expressed miRNAs and genes interact, elucidating some of the pathogenetic characteristics of PMF. Finally, IA was demonstrated a biologically relevant approach to identify aberrantly expressed miRNAs crucial to PMF pathogenesis, such as miR-494-3p and miR-155-5p, whose overexpressions are potentially involved in the abnormal megakaryopoiesis
Genomic landscape of megakaryopoiesis and platelet function defects
Full text linkMegakaryopoiesis is a complex, stepwise process that takes place largely in the bone marrow. At the apex of the hierarchy, hematopoietic stem cells undergo a number of lineage commitment decisions that ultimately lead to the production of polyploid megakaryocytes. On average, megakaryocytes release 1011 platelets per day into the blood that repair vascular injuries and prevent excessive bleeding. This differentiation process is tightly controlled by exogenous and endogenous factors, which have been the topics of intense research in the hematopoietic field. Indeed, a skewing of megakaryocyte commitment and differentiation may entail the onset of myeloproliferative neoplasms and other preleukemic disorders together with acute megakaryoblastic leukemia, whereas quantitative or qualitative defects in platelet production can lead to inherited platelet disorders. The recent advent of next-generation sequencing has prompted mapping of the genomic landscape of these conditions to provide an accurateview of the underlying lesions. The aims of this review are to introduce the physiological pathways of megakaryopoiesis and to present landmark studies on acquired and inherited disorders that target them. These studies have not only introduced a new era in the fields of molecular medicine and targeted therapies but may also provide us with a better understanding ofthemechanismsunderlying normalmegakaryopoiesis and thrombopoiesis that can informeffortsto create alternativesources of megakaryocytes and platelets
c-Myb supports erythropoiesis by transactivating KLF1 and LMO2 expression
No full textThe c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during
differentiation. c-myb is essential for the hematopoietic development, as c-myb-/- mice die at E15 due to failure of fetal
hepatic erythropoiesis. To gain further insights into the role of c-myb during the hematopoietic lineage commitment, we
studied the effects of c-Myb silencing in human CD34+ hematopoietic stem/progenitor cells. c-Myb silencing in CD34+
cells was performed by transfection of siRNAs using the Amaxa Nucleofector® Technology. In order to keep c-Myb
expression silenced for all the commitment phase of CD34+ cells, each sample was nucleofected 3 times, once a day.
Moreover, to exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample
transfected with the siRNAs targeting c-Myb, one sample electroporated without siRNAs and one transfected with a
non-targeting siRNA were performed. c-Myb silencing effects on CD34+ cells differentiation ability were studied by
methylcellulose and collagen-based clonogenic assays and by morphological and immunophenotypic analyses after
liquid culture. Furthermore, we investigated by microarray analysis the changes in gene expression induced by c-Myb
silencing. Methylcellulose assay revealed a remarkable increase of the percentage of monocyte (CFU-M) colonies and a
decrease of the erythroid ones (BFU-E) in c-Myb-silenced CD34+ cells. Moreover, collagen-based clonogenic assay
demonstrated that c-Myb silencing strongly enhances the megakaryocyte commitment of CD34+ cells. In agreement
with these data, flow cytometric analysis showed an increase in mono-macrophage and megakaryocyte fractions in cmyb-silenced
cells, while the erythroid population was strongly decreased. Morphological evaluation of May
Grunwald-Giemsa stained cytospins further supported the conclusion that c-myb silencing forces the CD34+ cells
commitment towards the macrophage and megakaryocyte lineages at the expense of the erythroid one. Gene expression
profiling of c-Myb silenced CD34+ cells enabled us to identify new putative targets which can account for c-Myb
knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb
binds to KLF1 and LMO2 promoters and transactivates their expression. Functional rescue experiments showed that the
retroviral vector-mediated overexpression of KLF1 and LMO2 transcription factors in c-Myb silenced cells is able to
rescue, at least in part, the impaired erythroid differentiation. Our data collectively demonstrate that c-Myb plays a
pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at
the expense of megakaryocyte diffentiation. In particular, we identified c-Myb-driven KLF1 and LMO2 transactivation
as the molecular mechanism through which c-Myb regulates erythroid versus megakaryocyte lineage fate decision
Characterisation and discovery of novel miRNAs and moRNAs in JAK2V617F mutated SET2 cells
Full text linkIn order to gain insights into a possible role of microRNAs in myeloproliferative neoplasms, we performed short RNAs massive sequencing and extensive bioinformatic analysis in the JAK2V617F mutated SET2 cell line. Overall, 652 known mature miRNAs were detected, of which 21 were highly expressed, thus being responsible of most of miRNA-mediated gene repression. microRNA putative targets were enriched in specific signaling pathways, providing information about cell activities under massive post-transcriptional regulation. The majority of miRNAs were mixtures of sequence variants, called isomiRs, mainly due to alternative, non-canonical processing of hairpin precursors. We also identified 78 novel miRNAs (miRNA*) derived from known hairpin precursors. Both major and minor (*) form of miRNAs were expressed concurrently from half of expressed hairpins, highlighting the relevance of miRNA* and the complexity of strand selection bias regulation. Finally, we discovered that SET2 cells express a number of miRNA-offset RNAs (moRNAs), short RNAs derived from genomic regions flanking mature miRNAs. We provide novel data about the possible origin of moRNAs, while their functional role remains to be elucidated. Overall, this study shed light on the complexity of microRNA-mediated gene regulation in SET2 cells and represents the basis for future studies in JAK2V617F mutated cellular models
miR-382-5p Controls Hematopoietic Stem Cell Differentiation Through the Downregulation of MXD1
No full textmicroRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional downregulation of hundreds of target genes through seed pairing in their 3' untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation, and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumors and hematological malignancies. miR-382-5p has been found to be upregulated in patients with myeloid neoplasms, but its role in normal hematopoiesis is still unknown. In this study, we demonstrated that miR-382-5p overexpression in CD34(+) hematopoietic stem/progenitor cells (HSPCs) leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones. Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (ie, FLI1, GATA2, MAF, MXD1, RUNX1, and SGK1). Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay. Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34(+) cells. Overall, our results demonstrated that miR-382-5p expression favors the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation. Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34(+) HSPCs
c-Myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression.
No full textThe c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-Myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity towards the macrophage and megakaryocyte lineages, while erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphological and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-Myb-silenced CD34+ cells identified the transcription factors KLF1 and LMO2 as putative targets which can account for c-Myb knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-Myb silencing, while only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-Myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression
No full textHistone deacetylase inhibitors represent a family of targeted anticancer compounds that are widely used against hematological malignancies. So far little is known about their effects on normal myelopoiesis. Therefore, in order to investigate the effect of histone deacetylase inhibitors on the myeloid commitment of hematopoietic stem/progenitor cells, we treated CD34(+) cells with valproic acid (VPA). Our results demonstrate that VPA treatment induces H4 histone acetylation and hampers cell cycle progression in CD34(+) cells sustaining high levels of CD34 protein expression. In addition, our data show that VPA treatment promotes erythrocyte and megakaryocyte differentiation. In fact, we demonstrate that VPA treatment is able to induce the expression of growth factor-independent protein 1B (GFI1B) and of mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), which are crucial regulators of erythrocyte and megakaryocyte differentiation, and that the up-regulation of these genes is mediated by the histone hyperacetylation at their promoter sites. Finally, we show that GFI1B inhibition impairs erythroid and megakaryocyte differentiation induced by VPA, while MLLT3 silencing inhibits megakaryocyte commitment only. As a whole, our data suggest that VPA sustains the expression of stemness-related markers in hematopoietic stem/progenitor cells and is able to interfere with hematopoietic lineage commitment by enhancing erythrocyte and megakaryocyte differentiation and by inhibiting the granulocyte and mono-macrophage maturation
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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