Nuclear microRNAs in normal hemopoiesis and cancer
Rasko and Wong Journal of Hematology & Oncology
Nuclear microRNAs in normal hemopoiesis and cancer
John E.J. Rasko 0 1 2
Justin J.-L. Wong 0 1 3 4
0 Sydney Medical School, University of Sydney , Camperdown, NSW 2050 , Australia
1 Gene & Stem Cell Therapy Program, Centenary Institute, University of Sydney , Camperdown 2050 , Australia
2 Cell and Molecular Therapies, Royal Prince Alfred Hospital , Camperdown 2050 , Australia
3 Locked Bag 6 , Newtown, NSW 2042 , Australia
4 Gene Regulation in Cancer Laboratory, Centenary Institute, University of Sydney , Camperdown, 2050 , Australia
Since the discovery of microRNAs (miRNAs) in the early 1990s, these small molecules have been increasingly recognized as key players in the regulation of critical biological processes. They have also been implicated in many diverse human diseases. The canonical function of miRNAs is to target the 3′ untranslated region (3′ UTR) of cytoplasmic messenger RNA to post-transcriptionally regulate mRNA and protein levels. It has now been shown that miRNAs can also bind to the promoter regions of genes or primary miRNA transcripts to regulate gene expression. Such observations have indicated the presence of miRNAs in the nucleus and implied additional noncanonical functions. Nevertheless, the role(s) of nuclear miRNAs in normal hemopoiesis and cancer remains elusive despite a burgeoning literature. Herein, we review current knowledge concerning the abundance and/or functions of nuclear miRNAs during blood cell development and cancer biology. We also discuss ongoing challenges in order to provoke further studies into identifying key roles for nuclear miRNAs in the development of other cell lineages and human cancers.
miRNAs; Hemopoiesis; Cancer; Gene regulation; Nuclear localization; Blood
Non-coding RNAs are RNA molecules that are not
translated into proteins but are nevertheless functional.
They include long non-coding RNAs, intronic RNAs,
circular RNAs, competing endogenous RNAs,
microRNAs (miRNAs) and Piwi-interacting RNAs that are
known to regulate gene expression at both the
transcriptional and post-transcriptional levels [1–5]. Of all
noncoding RNA species, miRNAs are best characterized in
terms of their biogenesis and functions.
miRNAs are approximately 22 nucleotides in length.
They play major roles in numerous biological processes
including cell differentiation, lineage specification,
reprogramming, immune response and the cell cycle [6–10].
Almost 30,000 miRNAs in 223 animal and plant species
have been annotated in the miRNA database, miRBase
(Release 21, June 2014). Even viruses, particularly
herpesviruses, encode miRNAs to enhance their replication
potential [11, 12]. The biological importance of miRNAs
is further highlighted by the deregulation of miRNA
expression in many diverse human diseases including
cardiovascular , neuronal [14, 15], inflammatory ,
dermatological , hepatological  and malignant
diseases [18–20]. Given their key roles in many normal
and disease-related processes, it is not surprising that
miRNAs are enthusiastically viewed as potential
druggable targets. To achieve this goal and anticipate side
effects, it is important to understand their mechanisms of
action and functions.
It is generally recognized that most mature miRNAs are
localized in the cytoplasm along with the four catalytic
Argonaute (Ago) proteins, where they contribute to the
RNA-induced silencing complex (RISC) [21–23]. It is via
this RISC complex that miRNAs regulate gene expression
by targeting messenger RNAs (mRNAs). The majority of
binding sites for miRNAs are within the 3′ untranslated
region (3′ UTR) of target mRNAs in animals, whereas they
are often within coding regions in plants . A mature
miRNA can bind to the 3′ UTR of a target mRNA based on
partial sequence complementarity between the two to
initiate one of several mechanisms to reduce mRNA and/or
protein levels. These mechanisms include repression of
translational elongation, impairment of translation initiation,
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and decapping and deadenylation of mRNA, which are
reviewed extensively elsewhere [25–27].
Notably, numerous nuclear miRNAs have also been
reported. These miRNAs may have diverse known or
unknown non-canonical functions. Herein, we discuss
how nuclear-localized miRNAs, although synthesized
through biogenesis pathways identical to cytoplasmic
miRNAs, could be shuttled back and retained in the
nucleus to exert functions that differ from canonical
miRNA actions. We focus especially on our current
knowledge concerning the distribution and roles of these
miRNAs in hemopoietic and cancer cells.
Evidence of nuclear-localized miRNAs
Systematic profiling analyses performed on nuclear and
cytoplasmic-fractionated RNA samples have concluded
that the majority of miRNAs are present in the nucleus
[28, 29]. These results indicate that most, if not all,
miRNAs have the capacity to shuttle between the nucleus
and cytoplasm. This finding is supported by the
localization of Ago proteins in the nucleus and a recent
discovery that a component of the RISC complexes,
TNRC6A, is a nuclear and cytoplasmic shuttling protein
that facilitates Ago nuclear transport. [30, 31] In vitro
knockdown of Importin 1 and 8 has also been shown to
reduce the nuclear localization of Ago proteins and/or
miRNAs, indicating that these transporter proteins
mediate the translocation of Ago/miRNAs into the nucleus
(see reviews by Liang et al.  and Roberts , for
details) [32, 33].
In addition to nuclear import mechanisms, there are
also miRNA-intrinsic aspects that facilitate nuclear
localization. The first evidence was provided by the
predominant localization of miR-29b in the nuclei of HeLa
and NIH3T3 cells, as directed by a hexanucleotide motif
(AGUGUU) at its 3′ terminus . A related miRNA,
miR-29a, lacked this hexanucleotide motif and is
enriched in the cytoplasm . Notably, a synthetic
siRNA harboring the miR-29a sequence engineered to
include the AGUGUU motif could be directed into the
nucleus, indicating the importance of this motif for
nuclear localization . Hwang and co-workers further
identified variants of this motif that is associated with
the nuclear localization of miRNAs, including
UGUGUU, ACUGUU, AGAGUU, AGUCUU, AGUGAU,
AGUGUA, AGNGUN . An independent study by
Jeffries and co-workers confirmed the presence of these
motifs in seven other nuclear-localized miRNAs:
miR30b, miR-30c, miR-19a, miR-374a, miR-374b,
miR-5905p and miR-193b .
However, these motifs alone may not be sufficient to
direct nuclear localization of miRNAs . For example,
miR-92b that possesses a hexanucleotide motif identical
to the nuclear-localized miR-29b has been reported to
be cytoplasmically enriched . A recent study also
failed to identify a relationship between nuclear or
cytoplasmic enrichment of miRNAs and their seed
sequences, suggesting that seed identity itself is insufficient
to determine their predominant localization . As yet
unknown sequence-independent factors may exist to
retain, detain or reimport specific miRNAs in the nucleus,
and they may also be cell-type specific.
Notwithstanding the lack of clarity regarding
mechanisms of nuclear miRNA localization, numerous studies
have demonstrated their enrichment and functions in
myriad mammalian cell types [37–45]. The functions of
these nuclear miRNAs include regulation of gene and
long non-coding RNA expression [37, 38, 46],
controlling the biogenesis of other miRNAs  and
finetuning the expression of mRNA expression in the
cytoplasm  (Fig. 1). Many of these functions are relevant
to hemopoiesis and cancer as outlined in the following
Nuclear-localized miRNAs in hemopoietic cells
Hemopoiesis is one of the first processes in which the
functions of miRNAs were elucidated [47, 48]. Lineage
commitment towards blood cell production begins with
the hemopoietic stem cell escaping quiescence and the
stepwise acquisition of specific myeloid or lymphoid
identities [49, 50]. The process involves gradual changes
in the expression patterns of hundreds of proteins,
including transcription factors [49, 50] and cell cycle
regulators , many of which are direct or indirect targets
More than a decade ago, three miRNAs, miR-181a,
miR-223 and miR-142 were recognized as key players in
myeloid and lymphoid cell differentiation . miR-181a
is preferentially expressed in the B-lineage, miR-223 in
the myeloid lineage and mir-142 in both B and myeloid
lineages . Remarkably, enforced expression of
miR181a led to a doubling of B cells while persistent
expression of miR-223 or miR-142 increased the number of T
cells by 30–40% . Subsequently, miR-181a has also
been found to be crucial for T cell development and
function, modulating T cell receptor signalling, in part
through its role in the downregulation of multiple
phosphatases including SHP2, PTPN22, DUSP5 and DUSP6
. Other confirmed targets of miR-181a include TCR,
CD69 and BCL-2, all of which are regulated during T cell
Given the substantial literature on the role of miRNAs
in hemopoiesis, studies that determined the cellular
localization of miRNAs in hemopoietic cells are
relatively few. Most studies have focused on the canonical
roles of miRNAs in the post-transcriptional regulation of
genes involved in hemopoiesis. The best examples of
nuclear miRNA functions have been reported in the
context of granulopoiesis (Table 1).
The myeloid specific miRNA, miR-223, has been well
characterized as playing an essential role in the control
of granulopoiesis. In the cytoplasm of myeloid
progenitors, miR-223 targets transcription factors MEF2C and
NF1A that usually promote myeloid progenitor cell
proliferation [54, 55]. The inhibition of cell proliferation in
myeloid progenitors coincides with their differentiation
into granulocytes. Notably, miR-223 also targets seed
sequences in the NF1A promoter to induce transcriptional
gene silencing via recruitment of the polycomb
repressive complex and a consequent increase in DNA
methylation levels . These data indicate that both nuclear
and cytoplasmic miR-223 work synergistically to silence
NF1A during granulopoiesis [42, 54].
Our group has recently reported the enrichment of
miRNAs including miR-690, miR-706 and miR-709 in
the nucleus of primary Lin−Sca+Kit+ hemopoietic stem/
progenitor cells, promyelocytes, myelocytes and
granulocytes . We and others have also found miR-690 and/
or miR-709 to be nuclear-enriched in various human
and mouse cell lines including MPRO, EL4, MEL, A20,
L929 and HEK-293T indicating that their functions may
be localized to the nucleus [41, 43]. One of these
miRNAs, miR-709, has been reported to bind with perfect
complementarity to pri-miR-15a and pri-miR-16-1 and
inhibit the biogenesis of these miRNAs . This finding
has established a previously unanticipated role of nuclear
miRNAs in regulating or fine-tuning the expression of
Our in silico analysis predicted many putative primary
miRNA (pri-miRNA) sequences that could be targeted
by miR-690, miR-706 or miR-709 with perfect
complementarity . Relevant to myelopoiesis, miR-706 is
predicted to bind perfectly to a hemopoeitic-specific
miR142-3p, which is known to play an essential role in
Table 1 Nuclear-localized miRNAs in normal hemopoiesis
miR-706 Myeloid (Lin−Sca+Kit+ hemopoietic stem/progenitor
cells, promyelocytes, myelocytes and granulocytes),
MPRO, EL4, MEL, A20
miR-690 Myeloid (Lin−Sca+Kit+ hemopoietic stem/progenitor
cells, promyelocytes, myelocytes and granulocytes),
MPRO, EL4, MEL, A20
miR-709 Myeloid (Lin−Sca+Kit+ hemopoietic stem/progenitor
cells, promyelocytes, myelocytes and granulocytes),
MPRO, EL4, MEL, A20
granulocyte homeostasis and maturation [43, 56].
Nevertheless, we were not able to demonstrate an increase of
pri-miR-142-3p processing following inhibition of
miR706 with a hairpin inhibitor against this miRNA .
Our result indicates the complexity of factors that
determine miRNA binding to its targets in the nucleus, which
are not likely to be dependent on sequences alone.
Importantly, we further reported that miRNAs may be
retained in the nucleus to fine-tune the expression of
mRNA targets . For example, miR-706 enrichment
in the nucleus is associated with decreased cytoplasmic
miR-706 expression. Consequently, the expression of its
targets such as the myeloid transcription factors, Stat1,
increases to promote myeloid differentiation [43, 57].
Nuclear-localized miRNAs in cancer
The nuclear localization of miRNAs in cancer cells is
well documented. Cancer cell lines including the 5-8F
nasopharyngeal carcinoma cells, the HCT116 colorectal
cancer cells and the THP-1 acute monocytic leukaemia
cells harbor hundreds of miRNAs that are enriched in
the nucleus of these cells [28, 29, 58]. The
nuclearspecific functions of many of these miRNAs remain
elusive, but they are likely to regulate or fine-tune the
expression of cancer-associated genes. For example,
miR10a, which is nuclear-localized in both the HCT116 and
THP-1 cell lines [29, 58], has been reported to inhibit
the transcription of Hoxd4 in the breast cancer cell lines,
MCF7 and MDA-MB-231 . Nuclear-localized
miR10a binds with near perfect complementarity to the
promoter of this tumor invasion and metastasis-promoting
gene to trigger its silencing via hypermethylation and
trimethylation of histone 3 lysine 27 at its promoter .
This example demonstrates that nuclear enrichment of
miR-10a may provide a therapeutic opportunity to
modulate gene expression relevant to cancer metastasis.
Fig. 1 Known roles of nuclear-localized miRNAs. a Regulation of gene expression by targeting gene promoters to activate or silence genes. b
Targeting and suppressing long non-coding RNA (lncRNA) function. c Perturbation of miRNA biogenesis via binding to primary miRNA (pri-mRNA)
transcripts. d Fine-tuning mRNA target expression through detention of miRNAs in the nucleus
Other nuclear-localized miRNAs have been reported
to promote transcriptional activation of oncogenes by
binding to their promoters. Examples include miR-483
that binds to the promoter of IGF2 to increase its
expression in Wilms’ tumors  and miR-558 that binds
to the promoter of heparanase (HPSE) to enhance its
expression, resulting in enhanced tumor growth in
neuroblastoma cells  (Table 2).
Nuclear-localized miRNAs can also protect against
tumorigenesis by promoting the activation of tumor
suppressor genes. For example, miR-373 binds to the
promoter of CDH1 to increase its expression in the
prostate cancer cell line, PC3 . miR-124
promotes the activation of P27, leading to G1 arrest in
myriad breast and ovarian cancer cell lines .
miR-205 induces the expression of the interleukin tumor
Activate IGF2 to enhance
Increase expression of CDH1 tumor
Activate interleukin tumor
suppressor genes, IL24 and IL32
Promote transcription of Ccnb1
Breast cancer cell lines: MCF7 and MDA-MB-231
Neuroblastoma cell lines: SK-N-SH, SK-N-AS, SH-SY5Y and SK-N-BE(2)
Prostate cancer cell line: PC3
Breast and ovarian cancer cell lines: MDA-MB-231, HeyA8, SKOV3.ip1,
BxPC-3 and L3.6pl, MIA PaCa 2, Panc1, U87, SNB19 and LN229
Prostate cancer cell lines: PC3, LNCaP and Du145
Mouse prostate adenocarcinoma cell line: TRAMP C1
Bladder cancer cell lines: T24 and EJ
Bladder cancer cell lines: T24 and EJ
Bladder cancer cell lines: T24 and EJ
Human neuroblastoma cell line: SH-SY5Y
suppressor genes, IL24 and IL32, by targeting specific
regions of their promoters . Additional examples are
provided in Table 2.
The mechanisms by which binding of miRNAs to gene
promoters results in transcriptional activation or
silencing have also been described. They include increased or
decreased levels of histone modifications associated with
gene activation [39, 45, 61], altered RNA Polymerase II
activity [38, 61], enhanced recruitment of transcription
factors  and inhibition of transcription factor binding
due to the presence of decoy miRNAs .
Besides coding genes, cancer-associated nuclear long
non-coding RNAs (lncRNAs), MALAT-1 and XIST, have
been reported as a target of miR-9 and miR-210,
respectively [63, 64]. The HOTAIR lncRNA, which localizes to
both the nucleus and cytoplasm, has also been reported
as a target of miR34a in prostate cancer cells .
However, it is unknown whether this interaction occurs in
the nucleus. The GENCODE Consortium has mapped
over 10,000 lncRNAs as putative miRNA targets, many
of which are nuclear-enriched . Further experimental
validations will no doubt identify more nuclear lncRNAs
as miRNA targets that are relevant to cancer.
Surprisingly, no report has previously reviewed the
role of nuclear miRNAs in haematological malignancies
despite the known roles of miRNAs as oncogenes or
tumor suppressor genes [18, 67]. miR-15a and miR-16-1
are established tumor suppressor miRNAs in chronic
lymphocytic leukaemia. Their biogenesis is known to be
regulated by the nuclear-localized miR-709 in liver cells
[41, 67]. Whether or not miR-709 fine-tunes the
expression of miR-15a and miR-16 in chronic lymphocytic
leukaemia remains to be determined. As described in the
previous section of this review, nuclear miR-223 has a
key role in normal granulopoiesis. A decrease in
miR223 expression level is associated with CEBPA-mutated
acute myeloid leukaemia . Inhibition of the miR-223
target gene, E2F1, by CEBPA is pivotal to prevent
leukemogenesis that results from E2F1-mediated
expression of the oncogene tribble (Trib) 2 gene [68, 69]. As
such both miR-223 and CEBPA regulate the expression
of E2F1 in myeloid cells under normal physiological
condition. Whether aberrant nuclear detention of miR-223
can occur to derepress E2F1 in CEBPA mutant
leukaemia demands further investigation.
Challenges in determining the functions of
The role of nuclear-localized miRNAs has been relatively
neglected as evidenced by the extensive literature on
miRNAs, including those relevant to hemopoiesis and
cancer. Indeed, the majority of studies have focused on
the action of miRNAs in post-transcriptional gene
regulation. Several reports have considered miRNAs’ role in
transcriptional regulation of specific mRNAs only when
these miRNAs did not act canonically via 3′ UTR
targeting [39, 61]. Others have focused on the transcriptional
regulation of specific genes by miRNAs because these
genes are known to be transcriptionally regulated by
other synthetic or endogenous double-stranded small
RNAs [37, 38]. In order to promote more rapid
elucidation of the nuclear-associated roles of thousands of
miRNAs, studies should specifically seek to identify nuclear
miRNAs and their functions, notwithstanding the
Most studies reporting nuclear-localized miRNAs have
utilized nuclear and cytoplasmic fractionation methods
to determine their localization. Inevitably, the difficulty
in obtaining a perfectly pure nuclear fraction has
provoked scepticism concerning the accuracy of reported
studies. Ensuring the removal of the cytoplasmic fraction
prior to collection of the nuclear fraction is crucial and
not always possible with some cells. It is also important
to perform validations to show the absence of
contaminating factors by western blot and RT-qPCR in
conjunction with microscopy-based detection of nuclear miRNA
labelled with fluorescence probes. These experimental
procedures are laborious and often require cell-type
specific optimizations to obtain reliable results. This is
particularly relevant for commercially available nuclear and
cytoplasmic extraction kits typically optimized for use
with commonly studied cell lines such as HeLa. In our
recent work, we undertook considerable optimization
steps beyond the manufacturer’s protocol to obtain good
quality nuclear and cytoplasmic fractions from primary
Upon identification of nuclear miRNAs, one next
logical step is to determine where they bind. Bioinformatic
tools have been developed to predict targets of miRNAs
within gene promoters or pri-miRNAs [70, 71]. Nearly
800,000 miRNA seed sequences match over 27,000
promoter sequences . miRNAs can also target gene
promoters through non-seed related complementarity ,
indicating that miRNA binding to promoter regions may
be more widespread than previously thought.
However, similar to the complexity of 3′ UTR
targeting by miRNAs, predicted miRNA-promoter/pri-miRNA
pairing often did not result in the expected functional
changes or occur in a cell-type specific manner [38, 43].
Thus, cause-effect experiments are necessary to confirm
that the binding of miRNAs to genomic sequences in
the nucleus results in functional consequences.
Experiments designed to reduce the expression of
miRNAs that localized specifically to the nucleus are
technically challenging. Indeed, our recent report shows
the dominant cytoplasmic localization of anti-miR-706
when transfected into myeloid cells . In a study that
reported the inhibition of miR-15a and miR-16
processing by miR-709 in mouse L929 liver cells, modest
upregulation of miR-15a and miR-16 (<2-fold) was
detected in anti-miR-709 transfected cells . It has not
been shown whether anti-miR-709 entered the nucleus
to inhibit miRNA processing. Thus, a direct role of
nuclear miR-709 in controlling the expression of other
miRNAs remains elusive.
A recent study has reported the utility of a
snoRNAbased vector (snoVector) that allows efficient nuclear
retention of RNA molecules processed from this vector
. RNA sequences such as lncRNAs, coding mRNAs
and precursor miRNAs (pre-miRNAs) can be inserted
into the snoVector. They can subsequently be processed
into functional RNAs via the endogenous snoRNA
processing machinery. RNA molecules expressed using
snoVector have been reported to be nuclear-enriched,
including those that are typically localized to the
cytoplasm . A similar vector (snoMEN) created via
manipulation of the human C/D small nuclear RNA
HBII180C has also been reported to target nuclear RNA .
It has been utilized to express interfering RNAs that
effectively reduced the expression of complementary
sequences including nuclear pre-mRNA and pri-miRNA
[74, 75]. Thus, it may be possible to use snoVector or
SnoMEN to constrain anti-miRNA sequences to the
nucleus. This step may facilitate functional characterization
following specific and efficient repression of nuclear
Nonetheless, it is important to recognize that miRNAs
have been reported to shuttle from the cytoplasm into
the nucleus. So, even if repression of specific nuclear
miRNAs is achieved, the observed functional loss may
be due to the overall depletion of cytoplasmic miRNAs
that are inhibited when they enter the nucleus. In such a
case, it would be very challenging to discern the specific
role of a given nuclear-localized miRNA. Optimal
experiment design should exclude changes in cytoplasmic
miRNA expression as a contributor to any phenotypic
alteration. Alternatively, despite experimental hurdles, it
is necessary to distinguish the function of any given
nuclear miRNA from its cytoplasmic counterpart.
The importance of miRNAs in hemopoiesis and cancer
through the post-transcriptional regulation of the
expression of relevant genes has been well-established.
In recent years, non-canonical roles for nuclear-localized
miRNAs have been uncovered. While there is evidence
that nuclear miRNAs regulate the transcription of
specific genes in hemopoietic and cancer cells, these
examples are relatively few in the context of the vast
published literature on miRNAs in these cell types. In this
review, we have described nuclear miRNAs that promote
granulopoieis and those that enhance the expression of
tumor suppressor genes and oncogenes. The roles of
nuclear miRNAs in the vast majority of myeloid and
lymphoid cells remain to be determined. Their roles in
cancer, including post-transcriptional silencing of tumor
suppressor genes, is as yet unreported. Therefore,
examination of the functions of these nuclear miRNAs should
prove to be a fruitful area for further research.
JEJR and JJ-LW received funding from the National Health and Medical
Research Council of Australia (Grant Nos. 1061906 to JEJR, 1080530 and
1128175 to JEJR and JJ-LW, and 1126306 to JJ-LW). JEJR is funded by the
Cancer Council of NSW, Cure the Future and an anonymous foundation.
JJLW holds a Fellowship from the Cancer Institute of NSW.
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