PU.1-Regulated Long Noncoding RNA lnc-MC Controls Human Monocyte/Macrophage Differentiation through Interaction with MicroRNA 199a-5p

Abstract

Long noncoding RNAs (lncRNAs) are emerging as important regulators in mammalian development, but little is known about their roles in monocyte/macrophage differentiation. Here we identified a long noncoding monocytic RNA (lnc-MC) that exhibits increased expression during monocyte/macrophage differentiation of THP-1 and HL-60 cells as well as CD34(+) hematopoietic stem/progenitor cells (HSPCs) and is transcriptionally activated by PU.1. Gain- and loss-of-function assays demonstrate that lnc-MC promotes monocyte/macrophage differentiation of THP-1 cells and CD34(+) HSPCs. Mechanistic investigation reveals that lnc-MC acts as a competing endogenous RNA to sequester microRNA 199a-5p (miR-199a-5p) and alleviate repression on the expression of activin A receptor type 1B (ACVR1B), an important regulator of monocyte/macrophage differentiation. We also noted a repressive effect of miR-199a-5p on lnc-MC expression and function, but PU.1-dominant downregulation of miR-199a-5p weakens the role of miR-199a-5p in the reciprocal regulation between miR-199a-5p and lnc-MC. Altogether, our work demonstrates that two PU.1-regulated noncoding RNAs, lnc-MC and miR-199a-5p, have opposing roles in monocyte/macrophage differentiation and that lnc-MC facilitates the differentiation process, enhancing the effect of PU.1, by soaking up miR-199a-5p and releasing ACVR1B expression. Thus, we reveal a novel regulatory mechanism, comprising PU.1, lnc-MC, miR-199a-5p, and ACVR1B, in monocyte/macrophage differentiation.

FIG 1

Bioinformatic analysis of lnc-MC to infer its potential role in myeloid differentiation. (A) The long noncoding RNA-Seq data for white blood cells are drawn from the supplemental file of the paper of Cabili et al. (30). The PU.1 ChIP-seq data pertaining to lncRNA were downloaded from ChIPBase. The data common to the two data sets were determined with a tool developed by a member of our lab, and the results are depicted as the intersection of two circles. (B) Expression spectrum of lnc-MC in 22 tissues and cell lines based on RNA-Seq data. lnc-MC expression in white blood cells is indicated by a vertical arrow. (C) UCSC Genome Browser screenshot showing the locations of PU.1 ChIP-seq, H3K27Ac ChIP-Seq, H3K4Me1 ChIP-seq, and RNA-Seq signals on the lnc-MC locus. (D) lnc-MC conservation among vertebrates was mapped using the UCSC Genome Browser. (E) The protein-coding potential of lnc-MC was analyzed using ORF Finder and the Coding Potential Assessment Tool.

FIG 2

lnc-MC mediates PMA-induced monocyte/macrophage differentiation. (A) qRT-PCR and semiquantitative PCR analyses of lnc-MC expression during PMA-induced monocyte/macrophage differentiation of THP-1 and HL-60 cells. GAPDH mRNA was used as an internal control. (B) qRT-PCR detection of lnc-MC and monocyte/macrophage differentiation marker CD11B, CD14, and CSF1R mRNAs in THP-1 cells infected with lenti-shlnc-MC or lenti-ctrl, followed by PMA induction for 50 h. Three independent experiments were performed, and data are means ± standard deviations. Asterisks indicate significant differences by Student's t test (*, P < 0.05; **, P < 0.01). (C) CD14 expression was evaluated by cytometric analysis in infected and PMA-induced cells. (Top) Results of a representative experiment. Red and black curves show results for untreated cells and anti-CD14 antibody-stained cells, respectively. (Bottom) Statistical analysis of three experiments. (D) May-Grünwald-Giemsa staining analysis of infected and PMA-induced cells. (Left) Results of a representative experiment. The arrows point to differentiated monocytes/macrophages. (Right) Statistical analysis of counts of differentiated monocytes/macrophages in five fields. (E) qRT-PCR detection of lnc-MC and CD11B, CD14, and CSF1R mRNAs in THP-1 cells infected with lenti-lnc-MC or lenti-ctrl, followed by PMA induction for 50 h. Three independent experiments were performed, and data are means ± standard deviations. (F) CD14 expression was evaluated by cytometric analysis in infected and PMA-induced cells. (Top) Results of a representative experiment. Red and black curves show results for untreated cells and anti-CD14 antibody-stained cells, respectively. (Bottom) Statistical analysis of three experiments. (G) May-Grünwald-Giemsa staining analysis of infected and PMA-induced cells. (Left) Results of a representative experiment. (Right) Statistical analysis of counts of differentiated monocytes/macrophages in five fields.

FIG 3

PU.1 modulates lnc-MC expression during monocyte/macrophage differentiation. (A) qRT-PCR analysis of lnc-MC expression and immunoblot analysis of PU.1 expression in THP-1 and HL-60 cells without (un) or with PMA induction for 48 h. The level of lnc-MC was normalized to the GAPDH mRNA level and is shown as fold expression relative to the levels in untreated cells. (B) Sketch map showing the putative PU.1 binding sites in lnc-MC gene loci. Boxes represent the putative binding sites of PU.1, and horizontal arrows flanking the boxes indicate the primers used for ChIP-PCR. The vertical arrow indicates the box confirmed by ChIP-PCR. The right-angle arrow indicates the transcriptional start site (TSS) and the direction of transcription. (C) ChIP-PCR assay of PU.1 binding. Only the box indicated by the vertical arrow in panel B was confirmed to be bound by PU.1. (D) Enhanced PU.1 binding to the site upstream of lnc-MC was detected in PMA-induced THP-1 and HL-60 cells. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by Student's t test. (E) Immunoblot analysis of PU.1 and qRT-PCR analysis of lnc-MC expression in THP-1 cells infected with lenti-shPU.1 or lenti-ctrl, followed by PMA induction. (F) Immunoblot analysis of PU.1 and qRT-PCR analysis of lnc-MC expression in THP-1 cells transfected with pcDNA6-PU.1 or pcDNA6, followed by PMA induction. (G) Rescue assays by infection with a combination of lenti-sh-PU.1 (or lenti-ctrl) and lenti-lnc-MC in CD34⁺ HSPCs. (Left) The expression levels of lnc-MC after infection were determined by qRT-PCR. (Center) CD14 expression was evaluated through by cytometry analysis. The gray and black curves indicate untreated cells and anti-CD14 antibody-stained cells, respectively. (Right) Statistical analysis of two independent CD14 expression experiments.

FIG 4

lnc-MC is negatively regulated by miR-199a-5p during monocyte/macrophage differentiation. (A) RNA FISH assay of lnc-MC in PMA-induced THP-1 cells. More than 20 cells were examined, and similar results were obtained. (B) Semiquantitative PCR detection of lnc-MC in the cytoplasmic (cyt) and nuclear (nuc) fractions of untreated (un) and PMA-induced THP-1 cells. GAPDH and U6 served as cytoplasmic and nuclear localization controls, respectively. (C) The interaction between lnc-MC and miR-199a-5p was predicted using the RNAhybrid Web tool. Partial sequences of lnc-MC (bottom) and miR-199a-5p (top) are shown. Numbers below the sequences indicate the positions of nucleotides relative to the transcriptional start site of lnc-MC. (D) qRT-PCR analysis of lnc-MC expression in THP-1 and HL-60 cells transfected with a miR-199a-5p mimic or a scrambled control (scr), followed by PMA induction for 48 h. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by Student's t test. (E) qRT-PCR analysis of lnc-MC expression at day 6 and day 13 of monocyte/macrophage induction culture of CD34⁺ HSPCs infected with lenti-miR-199a or lenti-ctrl. (F) miR-199a-5p sequence showing the predicted binding site (red) and lnc-MC sequences showing mutated or deleted nucleotides. wt, wild type; mut, mutant; del, deletant. (G) Luciferase reporter assays of 293TN cells cotransfected with each pMIR-Report-based construct and pcDNA6-miR-199a (or pcDNA6). Three independent experiments were performed, and data are means ± standard deviations. (H) Immunoprecipitation using an anti-Ago2 or anti-IgG antibody and extracts of PMA-induced THP-1 cells. (Top) Ago2 in immunoprecipitates was analyzed by immunoblotting. (Center) RNA levels in immunoprecipitates were determined by semiquantitative PCR. (Bottom) RNA levels in immunoprecipitates were determined by qRT-PCR. The levels of lnc-MC and β-actin are presented as fold enrichment in anti-Ago2 relative to anti-IgG immunoprecipitates. (I) Rescue assays in THP-1 cells. THP-1 cells were infected with lenti-miR-199a or lenti-ctrl. Twenty-four hours later, the cells were reinfected with lenti-lnc-MC or lenti-ctrl and were exposed to a medium with PMA for another 48 h. (Top) The expression levels of lnc-MC and miR-199a-5p were determined by qRT-PCR. (Bottom) (Left) CD14 expression was evaluated by flow cytometry analysis. The red and black curves indicate untreated cells and anti-CD14 antibody-stained cells, respectively. (Right) Statistical analysis of four independent experiments.

FIG 5

lnc-MC regulates miR-199a-5p expression and activity negatively. (A) miR-199a-5p sensor construct. (B) Luciferase reporter assays. 293TN cells were cotransfected with the miR-199a-5p sensor construct and either pcDNA6 or pcDNA6-199a alone or together with different doses of pcDNA6-lnc-MC or pcDNA6-lnc-MC-mut. Three independent experiments were performed, and data are means ± standard deviations. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by Student's t test. (C) qRT-PCR analysis of lnc-MC (top left) and miR-199a-5p (top right), and immunoblot analysis of ACVR1B expression (bottom), in THP-1 cells infected with lenti-shlnc-MC or lenti-ctrl, followed by PMA induction. (D) qRT-PCR analysis of lnc-MC (top left) and miR-199a-5p (top right), and immunoblot analysis of ACVR1B expression (bottom), in THP-1 cells infected with either lenti-lnc-MC, lenti-lnc-MC-mut, or lenti-ctrl, followed by PMA induction. (E) Schematic outline for identification of lnc-MC-associated RNA components by using the biotin-streptavidin system. (F) Relative abundances of lnc-MC and actin RNAs, as well as relative abundances of miR-199a-5p and miR-938, in lnc-MC probe and random probe pulldown assays are plotted as relative fold enrichment after normalization against GAPDH and U6 snRNA, respectively. Data are means ± standard deviations (n = 3).

FIG 6

Role and mechanism of lnc-MC in monocyte/macrophage differentiation of CD34⁺ HSPCs. (A) qRT-PCR analysis of lnc-MC expression during monocyte/macrophage differentiation of CD34⁺ HSPCs. D, day. (B) qRT-PCR analysis of lnc-MC and monocyte/macrophage differentiation marker CD14 and CSF1R mRNAs during induction culture of CD34⁺ HSPCs infected with lenti-lnc-MC or lenti-ctrl. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by Student's t test. (C) CD14 expression during induction culture of infected CD34⁺ HSPCs was evaluated by flow cytometry analysis. (Left) Histograms showing results of a representative experiment. The red and black curves indicate untreated cells and anti-CD14 antibody-stained cells, respectively. (Right) The results from two independent experiments were statistically analyzed and are presented as means ± standard deviations. (D) May-Grünwald-Giemsa staining analysis during induction culture of infected CD34⁺ HSPCs. (Left) Results of a representative experiment. (Right) Statistical analysis of counts of differentiated monocytes/macrophages in five fields. (E) qRT-PCR analysis of miR-199a-5p expression during induction culture of infected CD34⁺ HSPCs. (F) Immunoblot analysis of ACVR1B expression during induction culture of infected CD34⁺ HSPCs.

FIG 7

Schematic representation of the involvement of lnc-MC in the regulation of monocyte/macrophage differentiation. lnc-MC, transcriptionally activated by PU.1, facilitates monocyte/macrophage differentiation by acting as a ceRNA to sequester miR-199a-5p and release ACVR1B expression.