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 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.

Project description:Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ~1:200-500 and predisposes individuals to sudden death and heart failure. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and Results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured primary cardiac non-myocytes and analyzed the conditioned media by proteomics, we found that several ECM proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially expressed. We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac non-myocytes drives upregulation of ECM genes including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac non-myocytes. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac non-myocytes. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

Project description:The microenvironment of injured mucosa has important effects on intestinal stem cell self-renewal and reconstruction of epithelial barrier function in inflammatory bowel disease (IBD). However, the precise status of the interactions between intestinal epithelial cell (IEC) injury, particularly intestinal crypt absence, and microenvironment in IBD is not completely understood. We identified miR-494-3p as important for protection of colonic stemness in intestinal inflammation colonic organoid culture. A novel cytokine-cytokine receptor, EDA-A2/EDA2R, could suppress colonic stemness and epithelial repair during IBD. During intestinal inflammation, high level of LP macrophage-derived EDA-A2 inhibited the nuclear β-catenin/c-Myc axis and organoid growth by targeting EDA2R in colonic crypt stem cells. We further demonstrated that the pro-inflammatory cytokines IL-1β and IL-6 are capable of stimulating macrophages to release EDA-A2 during colitis. Secondly, we identified the cross-talk among IECs, colonic crypts, and lamina propria (LP) macrophages in miR-494-3p-mediated colitis. Furthermore, our study showed that miR-494-3p deficiency in IECs promoted LP macrophage recruitment and M1 activation in DSS-induced colitis mice. In addition, we identified miR-494-3p as critical to dampening IEC injury; specifically, miR-494-3p inhibited inflammation-induced IKKβ/NF-κB activation by targeting the IKKβ 3’UTR in IECs. As such, administration of adequate amounts of a miR-494-3p agomire attenuate colitis in vivo. Consistent with this inference, we showed that miR-494-3p levels were decreased in colonic crypts and serum in colitis mice, and loss of miR-494 potentiated the severity of colonic colitis. Our clinical data on the interactions between miR-494-3p levels in serum exosomes & colonic tissues and associated outcomes support the clinical relevance of miR-494-3p in IBD. The miR-494-3p agomir system, which we designed permits local delivery in vivo in this study, significantly ameliorated the severity of colonic colitis. Our findings no only uncover a miR-494-3p-mediated cross-talk mechanism by which inflamed colonic LP macrophages integrate signals from IECs to regulate colonic stemness and colonic epithelial repair/homeostasis. The miR-494-3p agomir may serve as a potential therapeutic approach in IBD.

Dataset Information

Gene expression profile (GEP) of CD34+ cells overexpressing miR-494-3p

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