Identification and Expression of Two microRNA Primary Transcripts, pri-miR-223 and pri-miR-10a, in Hematopoietic Cell Lines during Cytokine-Stimulated Differentiation.
Authors: Kosaka Nobuyoshi , Sugiura Keiichi , Nogawa Nami , Komatsu Norio , Kato Takashi 
Published: April 2020

Abstract
MicroRNAs (miRNAs) are small RNA molecules involved in various biological processes including cell differentiation, apoptosis, and oncogenesis. MiRNAs are generated from non-protein coding transcripts that are known as primary miRNA (pri-miRNA). Recently, some miRNAs were identified in hematopoietic cells. However, the relationship between pri-miRNA/miRNA expression and cytokine-induced cell proliferation/differentiation remains unclear. It has been reported that miR-223 is one of the miRNAs specifically expressed in murine myeloid lineages. We identified two splicing variants (672-nt and 437-nt) originated from its precursor pri-miR-223 (3256-nt). RT-PCR analysis by using oligo-dT primer and splice specific primer pair was performed to detect their expressions, and it revealed that pri-miR-223 was expressed in lung, brain, spleen and bone marrow but not in heart, liver or muscle. Notably each splicing variant displayed different tissue-specific expressions. Pri-miR-223 was more broadly expressed compared to the mature miR-223 expressed in bone marrow, spleen and thymus (Chen et al, Science, 2004), indicating that the generation of miR-223 is partly regulated by the miRNA processing pathway. In mouse promyelocytic NFS-60 cells, both pri-miR-223 splicing variants were expressed substantially when the cells were maintained in G-CSF (1ng/mL) exhibited granulocytic phenotype with myeloperoxidase expression, but the expression of the variants decreased on IL-3 (1ng/mL) stimulation: while the addition of G-CSF (10ng/mL) did not increase the expression of pri-miR-223. In other hematopoietic lineages, erythrocytic and megakaryocytic cells, the expressions of miRNAs had not been examined yet. As a first step to detect the miRNAs specific to erythrocytic and megakaryocytic cells, pri-miR-10a located within Hox gene clusters, was examined in the human cell lines established from the parent UT-7 cells, UT-7/GM, UT-7/EPO and UT-7/TPO. Their survival, growth and differentiation are known to be dependent on specific cytokine stimulations. Firstly, we identified the full-length sequence of human pri-miR-10a (482-nt) from EST database. RT-PCR analysis as with that for pri-miR-223 was then performed in each UT-7 cell line. Interestingly, pri-miR-10a was highly expressed in UT-7/EPO cells stimulated with erythropoietin but not in UT-7/TPO and UT-7/GM cells; though the expression of pri-miR-10a did not elevate by respective stimulation of erythropoietin (10U/mL), thrombopoietin (100ng/mL), or GM-CSF (10ng/mL) in UT-7/EPO, UT-7/TPO and UT-7/GM cells. These results showed that miR-223 and miR-10a expression was not regulated directly by cytokine stimulation, while their expression levels were altered by cytokine-dependent cell lineage commitment. The observations prompted us the hypothesis that lineage-specific miRNAs suppress the expression of their target genes that inhibit master transcription factors to direct gene silencing processes critical for differentiation. Since little information is available on transcription and processing of miRNAs specific to hematopoetic prolifiration and differentiation, our results provide a new approach to understand vital issues unsolved.