Similar Datasets
Project description:Gene expression profile (GEP) of CALR-overexpressing CD34+ cells and CALR-silenced CD34+ cells
| PRJNA382958 | ENA
Project description:The transcription factor cMyb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that cMyb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which cmyb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. mRNA and miRNA expression for each sample were profiled by Affymetrix GeneAtlas U219 strip array and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis.
2015-08-07 | GSE60299 | GEO
Project description:The transcription factor cMyb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that cMyb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which cmyb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. mRNA and miRNA expression for each sample were profiled by Affymetrix GeneAtlas U219 strip array and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. RNA from CD34+ HPCs transfected once/twice/3 times with c-myb-targeting/non targeting control siRNAs was collected for a set of 5 independent experiments.
2015-08-07 | E-GEOD-60299 | biostudies-arrayexpress
Project description:Primary Myelofibrosis (PMF) is a Philadelphia negative chronic myeloproliferative neoplasm characterized by bone marrow fibrosis, enhanced oxidative stress and high levels of serum pro-inflammatory cytokines. To identify genes and miRNAs potentially involved in PMF pathogenesis, we have previously carried out an integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem cells (HSPCs), which allowed us to identify miR-382-5p as up-regulated miRNA (Norfo R. et al, Blood 2014). Overexpression experiments in normal CD34+ cells have already demonstrated its central role in HSPC fate decision toward granulocyte lineage (Zini R. et al, Stem Cell Dev 2016). In this study, to further characterized the role of miR-382-5p in PMF pathogenesis, we performed a gene expression profile analysis in normal CD34+ HSPCs overexpressing miR-382-5p. Among the down-regulated genes upon miR-382-5p upregulation, we selected the anti-oxidant superoxide dismutase 2 (SOD2), depicted as the most favorable predicted target by TatgetScan 7.0. Firstly, luciferase reporter assay confirmed SOD2 as a real target of miR-382-5p. Furthermore, we showed that enforced miR-382-5p expression in CD34+ cells reduced SOD2 expression and activity, induced ROS accumulation, and oxidative DNA damages, as well as enhanced CD34+ cell proliferation. Afterwards, to confirm miR-382-5p as a key player in PMF pathogenesis, we performed inhibition experiments in PMF CD34+ cells revealing that miR-382-5p silencing restored SOD2 expression and activity, induced ROS disposal, decreased DNA oxidation and impaired cell proliferation.
2019-09-04 | GSE103464 | GEO
Project description:As recently reported by our group, we performed miRNA and gene expression profiling of CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 42 PMF patient samples compared with 31 healthy controls. Integrative analysis of these profiles by means of Ingenuity Pathway Analysis (IPA) allowed the identification of several aberrantly regulated miRNA-mRNA target pairs organized in interaction networks. In particular, our results highlighted the up-regulation of miR-494-3p in CD34+ cells from PMF patients (Norfo R et al, Blood, 2014). Interestingly, among the most upregulated miRNAs, miR-494-3p emerges as being associated to the highest number of downregulated target mRNAs. In order to understand the biological role of miR-494-3p during the hematopoietic commitment and differentiation, we overexpressed this miRNA in cord blood (CB) derived-CD34+ cells. Cells were electroporated with either miR-494-3p miRNA mimic (mimic miR-494) or a negative control mimic (mimic Neg CTR). qRT-PCR confirmed miR-494-3p overexpression 24h and 4 days after transfection (RQ ± SEM, 512.60 ± 137.37, p<.01, and 20.63 ± 3.03, p<.01, respectively). Immunophenotypic analysis of CD41 and CD42b megakaryocyte (MK) lineage differentiation markers, performed on serum-free megakaryocytic ultilineage culture at day 3, 5, 8, 10 and 12, demonstrated that miR-494-3p overexpression induces a significant increase in the MK fraction compared to control samples. Accordingly, morphological analysis of May-Grünwald-Giemsa stained cytospins revealed an expansion of megakaryocytic lineage in samples overexpressing miR-494-3p. These data were further confirmed by collagen-based clonogenic assays which showed a significant increase in the percentage of megakaryocytic colonies in miR-494-3p overexpressing samples compared with controls. In order to better characterize the molecular mechanisms underlying the effects of miR-494-3p on HSPCs differentiation, we performed gene expression analysis of miR-494-3p overexpressing cells 24 hours after the last nucleofection.
2017-06-12 | GSE85250 | GEO
Project description:In this work, we compared gene expression profile (GEP) of CD34+ cells transduced with the retroviral vector LCALRIDN with CD34+ cells transduced with the empty vector control LXIDN and of CD34+ cells treated with CALR siRNA with control cells transfected with a Non-Targeting siRNA In order to explore the role of CALR in the proliferation and differentiation of hematopoietic stem/progenitor cells (HPSCs), we studied the effects of CALR overexpression in Cord Blood (CB) CD34+ cells. Assessment of cell differentiation unveiled that CALR enforced expression is able to skew the haematopoietic commitment towards the MK and erythroid cell lineages. Conversely, CALR silencing induced a marked repression of MK and erythroid differentiation. In order to characterize the effects of CALR mutations on human hematopoietic cells at the molecular level, we analyzed the coding RNA profiles of CB CD34+ overexpressing CALR. This analysis identified a list of differentially expressed genes. Among upregulated genes, we found genes involved in platelet aggregation, inflammation and erythroid differentiation, potentially related to the characteristics of the disease.
2020-04-12 | GSE97807 | GEO
Project description:Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. Through a gene expression profile (GEP) study we recently highlighted the MAF upregulation in PMF versus healthy donor (HD) CD34+ hematopoietic progenitor cells (HPCs). To shed some light into the role of MAF in PMF pathogenesis, here we unravelled the effects of the overexpression of MAF in HPCs forcing its expression in HPCs. We showed that MAF overexpression favours the megakaryocyte and monocyte/macrophage commitment and leads to the abnormal expression of genes coding for proinflammatory and profibrotic mediators. Due to the key role of the above-mentioned processes in PMF pathogenesis, we selected a subset of genes coding for secreted molecules for further validation by quantitative enzyme-linked immunoassays. Noteworthy, our data unveiled a causal connection between the upregulation of MAF and the increased plasma levels of key proinflammatory/profibrotic mediators (IL8, CCL2, PLAUR and MMP9) in PMF patients. Similarly, the upregulation of MAF was responsible for the deranged expression of LGALS3 and SPP1, that are profibrotic mediators increased in PMF patients compared with HDs.
2017-12-31 | GSE85190 | GEO
Project description:The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Moreover, in order to identify the mRNA target through which hsa-miR-486-3p affects lineage fate decision, we profiled the mRNA changes in mimic transfected CD34+ HPC by means of Affymetrix GeneAtlas U219 strip array. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis.
2015-08-07 | GSE60298 | GEO
Project description:Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. Through a gene expression profile (GEP) study we recently highlighted the upregulationof miR-34a-5p in PMF versus healthy donor (HD) CD34+ hematopoietic progenitor cells (HPCs). To shed some light into the role of miR-34a-5p in PMF pathogenesis, here we unravelled the effects of the overexpression of miR-34a-5p in HPCs forcing its expression in HPCs. We showed that enforced expression of miR-34a-5p blocks proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF compared with HD HPCs. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression in HPCs lineage choice, by stimulating the megakaryocyte and monocyte/macrophage commitment.
2017-03-08 | GSE89156 | GEO
Project description:The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Moreover, in order to identify the mRNA target through which hsa-miR-486-3p affects lineage fate decision, we profiled the mRNA changes in mimic transfected CD34+ HPC by means of Affymetrix GeneAtlas U219 strip array. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. Gene expression profile (GEP) was performed on total RNA derived from three independent experiments at 24h after the last nucleofection.
2015-08-07 | E-GEOD-60298 | biostudies-arrayexpress