Wong Justin J L, Ritchie William, Gao Dadi, Lau Katherine A, Gonzalez Maria, Choudhary Anupma, Taft Ryan J, Rasko John E J, Holst Jeff
Gene & Stem Cell Therapy Program, Centenary Institute, Camperdown, Australia.
J Hematol Oncol. 2014 May 15;7:42. doi: 10.1186/1756-8722-7-42.
MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized.
We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets.
Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets.
Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.
微小RNA（miRNA）是细胞分化的协调因子，包括粒细胞生成。尽管许多miRNA的差异表达与粒细胞成熟有关，但从造血干细胞开始，对粒细胞生成所有阶段差异表达的miRNA及其细胞定位的分析尚不明确。
我们分别使用Taqman低密度芯片和Affymetrix芯片分析了粒细胞生成过程中的全细胞miRNA和mRNA表达。我们还进行了细胞核和细胞质分级分离，随后进行Taqman低密度芯片分析和/或定量PCR，以鉴定造血干/祖细胞、早幼粒细胞、中幼粒细胞、粒细胞和几种造血细胞系中核富集的miRNA。利用miRNA与其靶标对的表达之间的反相关性来确定假定的miRNA靶标。
对我们芯片数据的分析揭示了早幼粒细胞和粒细胞特有的差异表达miRNA的不同簇。虽然目前尚不清楚这些miRNA中的许多在粒细胞生成中的作用，但miRNA/mRNA靶标对表达的反相关性确定了一组新的靶基因。miRNA簇（包括let-7和miR-17-92家族成员）在造血干/祖细胞中下调，这可能使已知促进干细胞增殖和稳态的靶基因得以表达。此外，与其他主要富集在细胞质中的miRNA相比，发现四种miRNA（miR-709、miR-706、miR-690和miR-467a*）在髓系细胞和多种造血细胞系的细胞核中富集。miR-709和miR-706在整个粒细胞生成过程中都富集在细胞核中，并且在初级miRNA下游具有广泛互补性的假定结合位点。miR-467a*的核富集是造血干/祖细胞和早幼粒细胞特有的。这些miRNA在其他造血细胞系中也富集在细胞核中，在这些细胞系中，核隔离可能会微调细胞质mRNA靶标的表达。
总体而言，我们展示了以前未与造血分化相关的差异表达miRNA，并提供了miRNA核富集调控的进一步证据。对粒细胞发育中miRNA功能的进一步研究可能会揭示调控RNA生物学的基本方面以及核miRNA的作用。
