Argonaute 2 sustains the gene expression program driving human monocytic differentiation of acute myeloid leukemia cells.
ABSTRACT: MicroRNAs are key regulators of many biological processes, including cell differentiation. These small RNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Here, by using myeloid cell lines and primary blasts, we show that Ago2 has a key role in human monocytic cell fate determination and in LPS-induced inflammatory response of 1,25-dihydroxyvitamin D3 (D3)-treated myeloid cells. The silencing of Ago2 impairs the D3-dependent miR-17-5p/20a/106a, miR-125b and miR-155 downregulation, the accumulation of their translational targets AML1, VDR and C/EBP? and monocytic cell differentiation. Moreover, we show that Ago2 is recruited on miR-155 host gene promoter and on the upstream region of an overlapping antisense lncRNA, determining their epigenetic silencing, and miR-155 downregulation. These findings highlight Ago2 as a new factor in myeloid cell fate determination in acute myeloid leukemia cells.
Project description:MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.
Project description:BACKGROUND:Increased leukocyte adhesion to brain endothelial cells forming the blood-brain barrier (BBB) precedes extravasation into the central nervous system (CNS) in neuroinflammatory diseases such as multiple sclerosis (MS). Previously, we reported that microRNA-155 (miR-155) is up-regulated in MS and by inflammatory cytokines in human brain endothelium, with consequent modulation of endothelial paracellular permeability. Here, we investigated the role of endothelial miR-155 in leukocyte adhesion to the human cerebral microvascular endothelial cell line, hCMEC/D3, under shear forces mimicking blood flow in vivo. RESULTS:Using a gain- and loss-of-function approach, we show that miR-155 up-regulation increases leukocyte firm adhesion of both monocyte and T cells to hCMEC/D3 cells. Inhibition of endogenous endothelial miR-155 reduced monocytic and T cell firm adhesion to naïve and cytokines-induced human brain endothelium. Furthermore, this effect is partially associated with modulation of the endothelial cell adhesion molecules VCAM1 and ICAM1 by miR-155. CONCLUSIONS:Our results suggest that endothelial miR-155 contribute to the regulation of leukocyte adhesion at the inflamed BBB. Taken together with previous observations, brain endothelial miR-155 may constitute a potential molecular target for treatment of neuroinflammation diseases.
Project description:MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the "RNA-induced silencing complex" (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity.
Project description:Treatment of leukemia cells with 1,25-dihydroxyvitamin D3 may overcome their differentiation block and lead to the transition from myeloblasts to monocytes. To identify microRNA-mRNA networks relevant for myeloid differentiation, we profiled the expression of mRNAs and microRNAs associated to the low- and high-density ribosomal fractions in leukemic cells and in their differentiated monocytic counterpart. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular networks relevant for the monocyte cell fate determination, as for example the post-transcriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22-3p and let-7e-5p.
Project description:Blocks in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. 1,25-Dihydroxy-vitamin D3 (VitD3) arrests proliferation of AML cells and induces their differentiation into mature monocytes. In a previous study, we showed that miR-26a was induced upon VitD3-mediated monocytic differentiation. Here, we identify E2F7 as a novel target of miR-26a. We show that E2F7 significantly promotes cell cycle progression and inhibits monocytic differentiation of AML cells. We also demonstrate that E2F7 binds the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) (cyclin-dependent kinase inhibitor 1A) promoter repressing its expression. Moreover, interfering with E2F7 expression results in inhibition of c-Myc (v-myc myelocytomatosis viral oncogene homolog) transcriptional activity. This leads to the downregulation of c-Myc transcriptional target miR-17-92 cluster, whose expression has a well-defined role in contributing to block monocytic differentiation and sustain AML cell proliferation. Finally, we show that the expression of E2F7 is upregulated in primary blasts from AML patients. Thus, these findings indicate that the newly identified miR-26a target E2F7 might have an important role in monocytic differentiation and leukemogenesis.
Project description:microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases.
Project description:It has been reported that the two major types of RNA interference triggers, the classical Dicer-generated small RNAs (siRNAs), which function with all members of the Argonaute (Ago) protein family in mammals, and the Ago2-sliced small RNAs (sli-siRNAs), which function solely through Ago2, have similar potency in target cleavage and repression. Here, we show that sli-siRNAs are generally more potent than siRNAs in silencing mismatched targets. This phenomenon is usually more apparent in targets that have mismatched nucleotides in the 3' supplementary region than in targets with mismatches in the seed region. We demonstrate that Ago2 slicer activity is a major factor contributing to the greater silencing efficiency of sli-siRNA against mismatched targets and that participation of non-slicing Agos in silencing mismatched siRNA targets may dilute the slicing ability of Ago2. The difference in length of the mature guide RNA used in sli-RISCs and si-RISCs may also contribute to the observed difference in knockdown efficiency. Our data suggest that a sli-siRNA guide strand is likely to have substantially stronger off-target effects than a guide strand with the same sequence in a classical siRNA and that Dicer and non-slicing Agos may play pivotal roles in controlling siRNA target specificity.
Project description:Human cytomegalovirus (HCMV) resides in a latent form in hematopoietic progenitors and undifferentiated cells within the myeloid lineage. Maturation and differentiation along the myeloid lineage triggers lytic replication. Here, we used peripheral blood monocytes and the monocytic cell line THP-1 to investigate the effects of 1,25-dihydroxyvitamin D3 on HCMV replication. Interestingly, 1,25-dihydroxyvitamin D3 induces lytic replication marked by upregulation of HCMV gene expression and production of infectious virus. Moreover, we demonstrate that the effects of 1,25-dihydroxyvitamin D3 correlate with maturation/differentiation of the monocytes and not by directly stimulating the MIEP. These results are somewhat surprising as 1,25-dihydroxyvitamin D3 typically boosts immunity to bacteria and viruses rather than driving the infectious life cycle as it does for HCMV. Defining the signaling pathways kindled by 1,25-dihydroxyvitamin D3 will lead to a better understanding of the underlying molecular mechanisms that determine the fate of HCMV once it infects cells in the myeloid lineage.
Project description:High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-?B, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-?B to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-?B-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.
Project description:MicroRNAs (miRNAs) regulate cell proliferation, differentiation and death during development and postnatal life. The expression level of mature miRNAs results from complex molecular mechanisms, including the transcriptional regulation of their genes. MiR-223 is a hematopoietic-specific miRNA participating in regulatory signaling networks involving lineage-specific transcription factors (TFs). However, the transcriptional mechanisms governing its expression levels and its functional role in lineage fate decision of human hematopoietic progenitors (HPCs) have not yet been clarified. We found that in CD34(+)HPCs undergoing unilineage differentiation/maturation, miR-223 is upregulated more than 10-fold during granulopoiesis, 3-fold during monocytopoiesis and maintained at low levels during erythropoiesis. Chromatin immunoprecipitation and promoter luciferase assays showed that the lineage-specific expression level of mature miR-223 is controlled by the coordinated binding of TFs to their DNA-responsive elements located in 'distal' and 'proximal' regulatory regions of the miR-223 gene, differentially regulating the transcription of two primary transcripts (pri-miRs). All this drives myeloid progenitor maturation into specific lineages. Accordingly, modulation of miR-223 activity in CD34(+)HPCs and myeloid cell lines significantly affects their differentiation/maturation into erythroid, granulocytic and monocytic/macrophagic lineages. MiR-223 overexpression increases granulopoiesis and impairs erythroid and monocytic/macrophagic differentiation. Its knockdown, meanwhile, impairs granulopoiesis and facilitates erythropoiesis and monocytic/macrophagic differentiation. Overall, our data reveal that transcriptional pathways acting on the differential regulation of two pri-miR transcripts results in the fine-tuning of a single mature miRNA expression level, which dictates the lineage fate decision of hematopoietic myeloid progenitors.