Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells
Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells
Chengwu Zeng 0 1
Yan Xu 0 1
Ling Xu 0 1
Xibao Yu 0 1
Jingjing Cheng 0 1
Lijian Yang 1
Shaohua Chen 1
Yangqiu Li 0 1
0 Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University , Guangzhou 510632 , China
1 Institute of Hematology, Medical College, Jinan University , Guangzhou 510632 , China
Background: Acute promyelocytic leukemia (APL) is characterized by the reciprocal translocation t(15;17), which fuses PML with retinoic acid receptor alpha (RAR). Although PML-RAR is crucially important for pathogenesis and responsiveness to treatment, the molecular and cellular mechanisms by which PML-RAR 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-RAR 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-RAR. 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.
Acute promyelocytic leukemia (APL) is characterized by
an aberrant chromosomal translocation that fuses a
portion of the promyelocytic leukemia (PML) gene with the
retinoic acid receptor (RAR) gene , and subsequent
expression of the PML-RAR oncoprotein causes a block
at the promyelocytic differentiation stage. All-trans
retinoic acid (ATRA) has been successfully used as a leukemia
therapy to target the transcriptional repression mediated
by the PML-RAR fusion protein. The treatment of the
t(15;17) APL with ATRA induces the differentiation of
t(15;17) blasts and causes disease regression [2,3]
presumably through degradation of the chimeric protein encoded
by the PML-RAR oncogene [4-6].
Although PML-RAR is crucially important for
pathogenesis and responsiveness to treatment, the mechanism
by which PML-RAR exerts its oncogenic potential
remains unclear. Previous studies have proposed that
PMLRAR acts as a strong transcriptional repressor for target
genes by directly binding their promoter regions, which
are thought to include genes indispensable for myeloid
differentiation and apoptosis [7,8]. However, the number
of identified PML-RAR target genes is limited . Given
the structural and functional complexity of PML-RAR,
indirect effects of PML-RAR may play a significant role
in leukemic transformation. Indeed, PML-RAR may even
activate the expression a subset of genes [9,10]. These data
point to the necessity for addressing the issue of indirect
PML-RAR-mediated gene expression control.
Mammalian transcriptome studies have revealed large
numbers of long transcripts that have no protein-coding
potential. We previously demonstrated that microRNAs
play a significant role in the regulation of differentiation,
proliferation and apoptosis [11,12]. Compared with the
research progress of microRNAs, there are thousands of
longer transcripts whose functions are unknown.
Recently, several long non-coding RNAs (lncRNAs) have
been implicated in many types of cancers [13,14]. Our
preliminary data showed that NEAT1 is highly expressed
in the APL cell line NB4. NEAT1 (nuclear paraspeckle
assembly transcript 1) is a nuclear-restricted long
noncoding RNA that has two isoforms: 3.7 kb NEAT1_1
and 23 kb NEAT1_2 [15,16]. This non-coding RNA was
recently revealed to be an architectural component of a
subnuclear structure called the paraspeckle, which is
suggested to be involved in regulating gene expression
by retaining mRNAs for editing in the nucleus [16,17].
Although considerable progress has been made into the
paraspeckle composition, formation, and molecular
organization, the biological function of paraspeckles and
the role of the NEAT1 lncRNAs are incompletely
defined. In addition, it is not yet clear whether lncRNAs
are involved in APL pathogenesis. In this study, we
aimed to characterize the role and regulation of NEAT1
Patients and samples
A total of 43 peripheral blood samples including 31 APL
samples at diagnosis and 12 normal donors with informed
consent. All of the procedures were conducted according
to the guidelines of the Medical Ethics Committees of the
Health Bureau of the Guangdong Province of China, and
ethical approval was obtained from the Ethics Committee
of Medical School of Jinan University for this study.
Cell lines and cell cultures
NB4, NB4-R2 and U937-PR9 cell lines were kindly provided
by Dr. Yueqin Chen (Sun Yat-sen University, Guangzhou,
China) and cultured in RPMI 1640 containing 10% fetal
bovine serum. U937-PR9 contains a zinc-inducible
PMLRAR constructed from U937 . The cells were
cultured in a humidified atmosphere containing 5% CO2 at
37C. ATRA was purchased from Sigma-Aldrich and used
at the following final concentrations: 1 M ATRA (stock
10 mM in EtOH).Cell differentiation was assessed by
measuring the surface ITGAM/CD11b antigen expression
by flow cytometry analysis.
Quantitative real-time PCR analysis
qRT-PCR was performed to detect mature lncRNAs and
mRNA expression. Briefly, RNA was reverse-transcribed to
cDNA using High-Capacity cDNA Reverse Transcription
Kits(Applied Biosystems). ATCB served as internal control.
Primers were as follows: NEAT1 forward, 5-CTTCC
TCCCTTTAACTTATCCATTCAC-3; NEAT1 reverse,
forward, 5- CAGTTAGT
TTATCAGTTCTCCCATCCA3; NEAT1_2 reverse, 5-GTTGTTGTCGTCACCTTT
CAACTCT -3. qRT-PCR cycling program: 95C for
15 min, followed by 40 cycles at 95C for 10 s and 60C for
NB4 cells were transfected using the Neon Transfection
System (Invitrogen) with 100 pmol of oligonucleotides
in 10 l reactions. Transfection was performed as
described previously . The sequences of small
interfering RNA (siRNA) that specifically targets the
breakpoint region of PML-RAR were designed as previously
described . The following siRNA sequences targeting
the NEAT1 are as follows: 5-GUGAGAAGUUGCUUA
Cells (NB4 and U937-PR9) were washed twice in
phosphate-buffered saline (PBS) and lysed on ice for 30 min
in RIPA buffer. Protein extracts were separated in a
sodium dodecyl sulfate polyacrylamide electrophoresis
(SDS-PAGE) gel. The proteins were then transferred to a
polyvinylidenedifluoride (PVDF) membrane and probed
with anti-RAR (C-20; Santa Cruz Biotechnology) and
anti--actin (Sigma-Aldrich) antibodies.
Data were expressed as the mean SD of 3 independent
experiments. The significance of the differences between
groups was determined by a two-tailed Student t test. A
P-value <0.05 was considered significant.
NEAT1 express levels were downregulated in APL
Because NEAT1 has been proposed to control several
biological processes, including the stress response 
and cellular differentiation , we therefore initially
examined the expression level of NEAT1 in peripheral
blood mononuclear cells (PBMCs) from 31 cases with de
novo APL (13 males and 18 females with a median age
of 28.5 years and a range of 1752 years) expressing
the PML-RAR fusion gene, which is characterized by
leukemia blasts blocked at the promyelocyte stage of
differentiation. NEAT1 lncRNA is comprised of two
isoforms, NEAT1_1 (3.7 kb in humans) and NEAT1_2
(23 kb in humans ) (Figure 1A), and we used two primer
pairs that were designed as previously described to
quantify NEAT1 RNA isoforms by real-time quantitative
reverse transcription-PCR (qRT-PCR) . One primer
set recognizes both NEAT1_1 and NEAT1_2 (total
NEAT1), while the other recognizes only NEAT1_2.
qRT-PCR revealed that both NEAT1 and NEAT1_2 were
significantly decreased in APL patient samples compared
with normal granulocytes (Figure 1B), but not the
Malat1 lncRNA (Additional file 1: Figure S1A). This
result suggested that NEAT1 may be involved in APL
NEAT1 is suppressed by PML-RAR, and ATRA restores
The PML-RAR fusion protein is known to be the
initiating factor for APL development by transcriptionally
repressing retinoic acid and nonretinoic acid target
genes, we wondered whether NEAT1 downregulation was
a consequence of PML-RAR expression; thus, we
investigated the relationship between NEAT1 and PML-RAR.
To examine whether the expression of the NEAT1
lncRNAs is changed by PML-RAR expression, we used
a U937-derived cell line, U937-PR9, which contains
a zinc-inducible PMLRAR. U937-PR9 cells were
treated with 100 M ZnSO4 for the indicated time, and
PML-RAR was significantly upregulated (Additional
file 2: Figure S2A). Using this system, we found that
NEAT1 expression was significantly reduced in
PMLRAR-expressing cells (Figure 2A), but not in parental
U937 cells (Additional file 2: Figure S2B), indicating that
NEAT1 downregulation may be a consequence of
PMLRAR accumulation. Next, we used a small interfering
RNA (siRNA), which was designed according to a
previous report in which PML-RAR was effectively knocked
down by specifically targeting its breakpoint region .
The knockdown of PML-RAR by transfection with
si-PML-RAR was confirmed by qRT-PCR and western
blot (Additional file 2: Figure S2C). As expected, we found
that the expression of NEAT1 was increased by
PMLRAR knockdown (Figure 2B).
Because APL treatment leads to the clearance of
leukemia cells and loss of PML-RAR transcripts, we next
investigated the expression of NEAT1 and PML-RAR in
NB4 cells before and after ATRA treatment (1 M, stock:
10 mM in EtOH) to further establish the relationship
between NEAT1 and PML-RAR. Using qRT-PCR, the
expression level of total NEAT1 and NEAT1_2 in NB4
cells were rapidly increased upon treatment with ATRA.
Importantly, NEAT1 expression was not significantly
changed in ATRA-resistant NB4-R2 cells upon ATRA
treatment, excluding the possibility that the NEAT1
induction observed in NB4 cells represents a nonspecific
stress response to ATRA treatment rather than being
functional in differentiation (Figure 2C).
NEAT1 inhibition attenuates the myeloid differentiation
of APL cells
The above data show that the PML-RAR oncoprotein
represses the expression of NEAT1, and ATRA treatment
reverts the transcriptional repression mediated by the
PML-RAR fusion protein and increases NEAT1,
suggesting that NEAT1 may be involved in cell differentiation
and leukemogenesis. We then explored the functional role
of NEAT1 in the ATRA-induced myeloid differentiation
of APL cells. NEAT1 has been previously shown to be
effectively knocked down by siRNA [17,23], and it efficiently
attenuated the NEAT1 RNA level in this system compared
with control siRNA (Figure 3A). Transduced cells were
treated with ATRA, and after 48 h, the expression level
of CEBPB mRNA and a membrane antigen (ITGAM/
CD11b) associated with granulocytic cell differentiation
was measured . We found significantly reduced
CEBPB mRNA and ITGAM/CD11b levels in NB4 cells
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
Figure 2 lncRNA NEAT1 is repressed in cells expressing PML-RAR and upregulated in response to ATRA. (A) qRTPCR analysis of NEAT1
in U937-PR9 cells treated with 100 M ZnSO4 at the indicated time points. A time series of induction for the PML-RAR protein by ZnSO4 is
shown in Additional file 2: Figure S2A. (B) qRTPCR analysis of NEAT1 after knocking down PML-RAR. (C) NB4 and NB4-R2 cells were treated with
1 M 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.
transfected with si-NEAT1 compared with control cells
upon ATRA treatment (Figure 3B and C). These data
reveal a novel function for NEAT1 in myeloid differentiation.
In this study, we show that NEAT1 expression is
repressed by PML-RAR. In addition, we provide evidence
that NEAT1 expression is involved in the differentiation
of APL cells.
PML-RAR is a potent transcriptional repressor in
APL cells, and it blocks promyelocyte differentiation. An
interesting characteristic of this oncogenic protein is that
its transcriptional repression effects can be reverted with
pharmacologic doses of ATRA, resulting in the
reactivation of genes essential for definitive myeloid
differentiation [2,3,25]. Recently, lncRNAs have been shown to
be dysregulated in various cancers, and several lncRNAs
have been functionally linked to cancer and cell
differentiation [13,15]. In this study, our findings of particular
low NEAT1 levels in APL cells indicate that NEAT1
transcription is further repressed by PML-RAR.Indeed,
we found that NEAT1 was repressed by PML-RAR,
and this repression could be relieved by ATRA. These
results suggest that NEAT1 expression is regulated by
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 qRTPCR 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 M). 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.
PML-RAR. Unlike ATRA, arsenic trioxide induces
limited transcriptional changes of NEAT1 in APL cells
(Additional file 3: Figure S3A). This result may due to
arsenic trioxide induces significant cell death but only
very limited differentiation [26,27], and upregulation of
NEAT1 in ATRA-treated APL cells is a consequence of
the relief of PML-RAR-mediated transcriptional
repression. Notably, previous studies have indicated
PMLRAR is able to interact with many other transcription
factors, such as PU.1, providing the potential for the
oncoprotein to target genes primarily regulated by other
transcription factors [7,28]. However, due to the
structural and functional complexity of PML-RAR, whether
this regulation is by direct DNA binding or by other
transcription factors requires further investigation. More
importantly, this lncRNA is involved in the differentiation
of APL cells, suggesting an interesting characteristic for
the PML-RAR oncogenic protein in the regulation of
gene expression by NEAT1. The mammalian nucleus is
highly organized and contains several membraneless
subcompartment nuclear bodies, including nucleoli,
paraspeckles, PML bodies and speckles, which are thought to
be involved in gene regulation . NEAT1 is a critical
component of the paraspeckle structure, and paraspeckles
have been proposed to be involved in the regulation of
gene expression through the control of the nuclear
retention of mRNAs containing long inverted repeats, which
are capable of forming intramolecular double-stranded
RNAs subject to adenosine-to-inosine editing . More
recently, NEAT1 was also shown to be involved in
transcriptional regulation by sequestrating a transcriptional
regulator . Previous studies have demonstrated that
PML-RAR was capable of promoting leukemic
transformation by impairing the formation of functional PML
nuclear bodies . Thus, PML-RAR may influence the
nuclear retention of structured mRNAs or gene
transcription, which is indispensable for myeloid differentiation,
through NEAT1. This finding is the first evidence that a
lncRNA cooperates with a fusion protein and plays a
critical role in the response to treatment. However, the
underlying molecular mechanisms of NEAT1 and potential
roles of paraspeckles in APL require further investigation.
In conclusion, we report abnormally decreased
expression of the nuclear long noncoding RNA NEAT1, which
is responsible for the differentiation block in blast cells
in APL. These findings provide a more comprehensive
understanding of APL pathogenesis. Because NEAT1
plays an important role in the posttranscriptional and
transcriptional regulation of gene expression and we
found that the expression of this nuclear long
noncoding RNA was regulated by PML-RAR, it remains an
interesting open question whether subsets of genes
essential for myeloid differentiation are also modulated
by perturbation of NEAT1 expression.
Taken together, these results are the first to assign a
biological function to the nuclear long noncoding RNA
NEAT1 in the myeloid differentiation of APL cells and
may lead to a fuller understanding of the molecular
events leading to APL.
Additional file 1: Figure S1. Analysis of Malat1 expression in APL
primary patient samples. (A) qRTPCR and western blot analysis of Malat1
expression in granulocytes from healthy donors (Granulo, n =12)
compared with primary APL cells (n =31). 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.
Additional file 2: Figure S2. NEAT1 is suppressed by PML-RAR.
(A) qRTPCR and western blot analysis of PML-RAR in U937-PR9 cells
induced with 100 M ZnSO4. (B) qRTPCR analysis of NEAT1 in U937 cells
treated with 100 M ZnSO4 at the indicated time points. NEAT1 was
normalized to the housekeeping gene ACTB. The panels show the
mean SD of a representative experiment performed in triplicate.
(C) qRTPCR analysis and western blots were performed to detect
PML-RAR in NB4 cells after transfection with si-PML-RAR (Students t test
was used to calculate the p value).
Additional file 3: Figure S3. Analysis of NEAT1 expression. (A) qRTPCR
analysis of NEAT1 in NB4 cells treated with 2 M arsenic trioxide at the
indicated time points. The ACTB level is shown as a loading control.
CWZ designed and performed the research, analyzed data and wrote the
manuscript. YX, LX, XBY, JJC, LJY and SHC performed the research and
analyzed data. YQL designed the research and helped draft the manuscript.
All authors read and approved the final manuscript.
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