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Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells.

ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

SUBMITTER: Papasavva PL

PROVIDER: S-EPMC8037168 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) crucial for post-transcriptional and translational regulation of cellular and developmental pathways. Study of miRNAs in erythropoiesis thus elucidates the process and facilitates diagnosis and therapy development for related disorders. Here, we used high-throughput DNA Nanoball small RNA sequencing to characterise sncRNAs, and in particular miRNAs, comprehensively in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and on two influential erythroid cell lines with adult and fetal expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential expression (DE) to erythroid differentiation, cell type, and fetal and adult hemoglobin expression patterns. Sequencing results were validated by reverse-transcription quantitative PCR (RT-qPCR) of selected DE miRNAs (miR-451a, miR-182-5p, miR-146a-5p, miR-221-3p, miR-222-3p) and indicated overall common erythroid differentiation signatures in primary cells and cell lines, in terminal stages of erythropoiesis characterized by decreased complexity of global miRNA expression and reciprocally increased expression of individual lineage-specific miRNAs. Interestingly, against a backdrop of evident clustering and thus similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted readily interpretable discrepancies between both cell types. Moreover, differential expression analysis between the closely related HUDEP-2 and HUDEP-1 cells provided pointers to miRNAs, transcription factors (TFs), target genes, and pathways mechanistically linked to globin switching. In resulting TF-miRNA co-regulatory networks, TFs known to be implicated in hemoglobin switching, including the major γ-globin repressors BCL11A and ZBTB7A, were targeted by many co-regulated miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

Project description:IntroductionIn human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses.MethodsScleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods.ResultsHuman sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10(11)). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples.ConclusionFor the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.

Project description:BACKGROUND:Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by "bulk" assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. RESULTS:To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: "Pre-MEP," enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; "E-MEP," strongly biased towards erythroid differentiation; and "MK-MEP," a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. CONCLUSIONS:Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias.

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Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells.

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