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MicroRNA expression during murine fetal liver megakaryocyte differentiation

ABSTRACT: MicroRNAs are small non-coding RNAs that regulate cellular development by interfering with mRNA stability and translation. We defined the kinetics of global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were upregulated and 81 were downregulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were upregulated most strongly in megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly upregulated during mouse and human megakaryopoiesis, but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously.

ORGANISM(S): Rattus norvegicus Human gammaherpesvirus 8 Betapolyomavirus hominis Mus musculus Human alphaherpesvirus 1 JC polyomavirus human gammaherpesvirus 4 Betapolyomavirus macacae Homo sapiens Human immunodeficiency virus 1 Human betaherpesvirus 5

PROVIDER: GSE22480 | GEO | 2010/12/07

SECONDARY ACCESSION(S): PRJNA128689

REPOSITORIES: GEO

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Project description:MicroRNAs are small non-coding RNAs that regulate cellular development by interfering with mRNA stability and translation. We defined the kinetics of global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were upregulated and 81 were downregulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were upregulated most strongly in megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly upregulated during mouse and human megakaryopoiesis, but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously. Exiqon locked nucleic acid (LNA) microarrays were used to compare microRNA expression in starting populations (ter 119- progenitors) and purified megakaryocytes. Day13.5-14.5 murine fetal livers (strain CD1) were depleted of erythroid cells and cultured with thrombopoietin to generate megakaryocytes. Each total RNA sample (0.5 Î¼g per reaction) was labeled with Hy3 and Hy5 dyes using the Exiqon Power Labeling Kit and automated microarray hybridizations and washes were performed on a Tecan HS4800 station with 20 hr hybridization at 56ÂºC. Dye-swap pairs of three replicate experiments comparing Ter119- fetal liver cells vs. BSA-purified megakaryocytes were co-hybridized to six arrays. The geometric average of the 532 and 635 measurements after normalization was determined for each sample.

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.

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 with c-myb-targeting/non targeting control (NegCTR) synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments.

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.

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MicroRNA expression during murine fetal liver megakaryocyte differentiation

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