Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells - PubMed (original) (raw)

Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells

Chengwu Zeng et al. BMC Cancer. 2014.

Abstract

Background: Acute promyelocytic leukemia (APL) is characterized by the reciprocal translocation t(15;17), which fuses PML with retinoic acid receptor alpha (RARα). Although PML-RARα is crucially important for pathogenesis and responsiveness to treatment, the molecular and cellular mechanisms by which PML-RARα exerts its oncogenic potential have not been fully elucidated. Recent reports have suggested that long non-coding RNAs (lncRNAs) contribute to the precise control of gene expression and are involved in human diseases. Little is known about the role of lncRNA in APL.

Methods: We analyzed NEAT1 expression in APL samples and cell lines by real-time quantitative reverse transcription-PCR (qRT-PCR). The expression of PML-RARα was measured by Western blot. Cell differentiation was assessed by measuring the surface CD11b antigen expression by flow cytometry analysis.

Results: We found that nuclear enriched abundant transcript 1 (NEAT1), a lncRNA essential for the formation of nuclear body paraspeckles, is significantly repressed in de novo APL samples compared with those of healthy donors. We further provide evidence that NEAT1 expression was repressed by PML-RARα. Furthermore, significant NEAT1 upregulation was observed during all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Finally, we demonstrate the importance of NEAT1 in myeloid differentiation. We show that reduction of NEAT1 by small interfering RNA (siRNA) blocks ATRA-induced differentiation.

Conclusions: Our results indicate that reduced expression of the nuclear long noncoding RNA NEAT1 may play a role in the myeloid differentiation of APL cells.

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Figures

Figure 1

Figure 1

The lncRNA NEAT1 is significantly down-regulated in APL primary patient samples. (A) The NEAT1 isoforms are shown schematically. The black boxes indicate the position of sequences amplified by qRT-PCR. (B) Comparison of NEAT1 expression in granulocytes from healthy donors (Granulo, n =12) compared with primary APL cells (n =31). The expression levels of the NEAT1 isoforms were evaluated by qRT-PCR. Malat1 lncRNA served as negative control and shown in Additional file 1: Figure S1A. Measured cycle threshold (Ct) values represent log2 expression values. The values were normalized to the expression level of the housekeeping gene ACTB. Each data point represents 1 patient sample.

Figure 2

Figure 2

lncRNA NEAT1 is repressed in cells expressing PML-RARα and upregulated in response to ATRA. (A) qRT–PCR analysis of NEAT1 in U937-PR9 cells treated with 100 μM ZnSO4 at the indicated time points. A time series of induction for the PML-RARα protein by ZnSO4 is shown in Additional file 2: Figure S2A. (B) qRT–PCR analysis of NEAT1 after knocking down PML-RARα. (C) NB4 and NB4-R2 cells were treated with 1 μM ATRA. NEAT1 was measured by qRT-PCR and normalized to the housekeeping gene ACTB. The panels show the mean ± SD of a representative experiment performed in triplicate.

Figure 3

Figure 3

Knocking down NEAT1 impairs neutrophil differentiation in APL cells. (A) 48 hrs after transfection, the knockdown efficiency was confirmed by qRT-PCR. (B) CEBPB mRNA levels were measured by qRT–PCR and are given as n-fold changes compared with untreated cells and normalized to the housekeeping gene ACTB. (C) Flow cytometry analysis of ITGAM/CD11b surface expression of control and si-NEAT1 cells upon 48 h of ATRA-treatment (1 μM). CD11b expression was measured by flow cytometry and values were normalized to untreated control cells. Data are shown as the mean ± SD of three separate experiments.

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Pre-publication history
    1. The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/693/prepub

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