Download miR-34a contributes to megakaryocytic differentiation of K562 cells

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PLATELETS AND THROMBOPOIESIS
miR-34a contributes to megakaryocytic differentiation of K562 cells
independently of p53
Francisco Navarro,1 David Gutman,1 Eti Meire,2 Mario Cáceres,3 Isidore Rigoutsos,4 Zvi Bentwich,2 and Judy Lieberman1
1Immune
Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, MA; 2Rosetta Genomics, Rehovot, Israel; 3Genes and Disease
Program, Centre for Genomic Regulation, Barcelona, Spain; and 4Bioinformatics and Pattern Discovery Group, IBM Thomas J. Watson Research Center,
Yorktown Heights, NY
The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a
is strongly up-regulated during phorbol
ester–induced megakaryocyte differentiation, but not during hemin-induced erythrocyte differentiation. Enforced expression of
miR-34a in K562 cells inhibits cell proliferation, induces cell-cycle arrest in G1 phase,
and promotes megakaryocyte differentiation as measured by CD41 induction. miR34a expression is also up-regulated during
thrombopoietin-induced differentiation of
CD34ⴙ hematopoietic precursors, and its
enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates
expression of MYB, facilitating megakaryocyte differentiation, and of CDK4 and CDK6,
to inhibit the G1/S transition. However, these
miR-34a target genes are down-regulated
rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This
suggests that miR-34a is not responsible for
the initial down-regulation but may contribute to maintaining their suppression later
on. Previous studies have implicated miR34a as a tumor suppressor gene whose
transcription is activated by p53. However,
in p53-null K562 cells, phorbol esters induce
miR-34a expression independently of p53
by activating an alternative phorbol esterresponsive promoter to produce a longer
pri-miR-34a transcript. (Blood. 2009;114:
2181-2192)
Introduction
microRNAs (miRNA), small noncoding RNAs that suppress
expression of genes bearing partially complementary sequences,
participate in regulating hematopoietic cell differentiation and
leukemogenesis.1 miRNA expression changes dramatically during
cell differentiation. In some cases, alterations in a single miRNA
can have a significant impact on cell-lineage commitment and
survival. A general requirement for miRNAs during T-cell development was demonstrated by studies in mice selectively deleted of
Dicer-1 in the thymus, which show impaired CD8 T-cell development and defects in T-helper cell differentiation and function.2,3
miR-181a regulates T-cell differentiation to effector cells by
inhibiting the expression of multiple phosphatases, and miR-155
regulates regulatory T cell–lineage commitment in part by inhibiting SOCS1 expression.4,5 miR-223 regulates granulocytic differentiation through translational inhibition of the transcription factor
NFI-A.6 miR-150, whose expression is largely restricted to mature
lymphocytes, facilitates B-cell development and megakaryocyte
differentiation of megakaryocyte-erythrocyte progenitors (MEPs)
by regulating MYB expression.7,8
miRNAs have also been implicated in leukemogenesis and
cancer more generally. A high proportion of miRNA genes are
encoded in cancer-associated regions associated with loss of
heterozygosity, gene amplification, common breakpoint regions,
and fragile sites.9 miRNAs can function as either tumor suppressors
or oncogenes. Oncogenic miRNAs in hematologic malignancies
include miR-155 and the miRNA cluster miR-17-92, which are
both amplified in different types of B-cell lymphoma.1 The
miR-15a/16-1 cluster, encoded in a region of chromosome 13 that
is frequently deleted in B-cell chronic lymphocytic leukemia, is a
tumor suppressor miRNA.10,11
miR-34a is another example of a tumor suppressor. miR-34a is
encoded within the chromosome region 1p36 whose loss has been
associated with glioma, neuroblastoma, pancreatic cancer, and
chronic myelogenous leukemia.12-15 miR-34a is part of the p53
regulatory network.16 miR-34a transcription is directly activated by
p53, and in turn miR-34a regulates the expression of some p53
target genes.15,17,18 In support of its role as a tumor suppressor,
miR-34a has been shown to regulate genes involved in cell-cycle
regulation and apoptosis, including CDK4, CDK6, CCND1, E2F3,
and SIRT1.14,17,19,20
The human erythroleukemia cell line K562, derived from a
chronic myelogenous leukemia patient, resembles a bipotent
MEP.21 Phorbol esters, such as 12-O-tetradecanoyl-phorbol-13acetate (TPA), induce megakaryocyte (MK) differentiation, whereas
hemin, sodium butyrate, or Ara-C induces differentiation to
erythrocytes.22 TPA-induced MK differentiation of leukemic K562
cells is accompanied by characteristic changes in cell morphology,
cell adhesion, cell-cycle arrest, endomitosis, and expression of MK
lineage–specific markers, such as platelet-derived growth factor
and the ␣IIb␤3 (CD41/CD61) and ␣2␤1 (CD49b) integrins.22 We
used this cellular model to investigate the role of miRNAs in MK
differentiation. We found that miR-34a is the most highly upregulated miRNA during TPA-induced MK differentiation of
leukemic K562 cells and is part of a miRNA signature of
up-regulated miRNAs specific to the MK lineage. miR-34a contributes to MK differentiation by inhibiting cell proliferation and
Submitted February 11, 2009; accepted June 28, 2009. Prepublished online as
Blood First Edition paper, July 7, 2009; DOI 10.1182/blood-2009-02-205062.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2009 by The American Society of Hematology
BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
NAVARRO et al
inducing expression of MK-specific markers, at least in part,
through direct regulation of its known targets CDK4 and CDK6 and
the newly identified target MYB. miR-34a also enhances MK
differentiation of CD34⫹ hematopoietic progenitor cells. We also
show that miR-34a expression in K562 cells is p53 independent.
An alternative promoter drives expression of a longer pri-miR-34a
transcript in response to phorbol esters.
Methods
Cell culture and reagents
Human erythroleukemia K562 cells (ATCC) were grown in RPMI-1640
medium supplemented with 10% fetal calf serum and penicillin/
streptomycin (Invitrogen). For MK differentiation, cells were treated with
10 nM TPA (Sigma-Aldrich). For erythrocyte differentiation, 100 ␮g/mL
hemin (Sigma-Aldrich) was added to the medium for the duration of the
experiment. Benzidine staining detected hemoglobin-positive cells. Frozen
human umbilical cord blood CD34⫹ cells were grown in StemSpan H3000
Defined Medium in the presence of the cytokine cocktail CC200 (containing 50 ng/mL recombinant human [rh] thrombopoietin [TPO], 50 ng/mL rh
stem cell factor [SCF], and 10 ng/mL rh interleukin-3 [IL-3]; StemCell
Technologies).
miRNA and gene microarrays
For miRNA analysis, small RNA libraries were prepared as described
previously.23 cRNA was generated and labeled with Cy3-CTP or Cy5-CTP
using the Low-Input Linear Amplification Kit (Agilent Technologies).
Samples were hybridized on custom oligonucleotide microarrays (Agilent
Technologies) containing probes for 286 annotated miRNAs.24 Hybridized
microarrays were scanned using the Agilent LP2 DNA Microarray Scanner,
and microarray images were analyzed using Feature Extraction Software
(Version 7.1.1; Agilent). mRNA microarray analysis was performed using
15 ␮g total RNA applied to Human Genome U133 Plus 2.0 Arrays
(Affymetrix) at the Microarray Core Facility at the Dana-Farber/Harvard
Cancer Center core facility. Data were analyzed using Microarray Suite 5.0
software (Affymetrix). Microarray data are available at the public databases
ArrayExpress (TPA-induced MK differentiation experiment; accession no.
E-MEXP-2213) and Gene Expression Omnibus (miR-34a overexpression
experiment; accession no. GSE16674).
miRNA mimics and transfections
K562 cells were transfected by nucleofection (Amaxa) with 5 ␮g plasmid
DNA or 3 ␮g miRNA mimic or control mimic (Dharmacon) as per the
manufacturer’s protocol.
Colony assays and lentivirus infections
A total of 5 ⫻ 104 human cord blood CD34⫹ cells (StemCell Technologies)
were infected overnight with high-titer miRNA-expressing lentiviruses.
Cells were washed the next day with phosphate-buffered saline and plated
for colony-forming units–megakaryocyte (CFU-MK) assays using the
MegaCult-C kit (StemCell Technologies) following the manufacturer’s
instructions. After incubation at 37°C for 12 days, MK colonies were scored
after CD41 staining.
Quantitative RT-PCR
Analysis of mature miRNA expression relative to U6 snRNA was performed using miRNA-specific quantitative reverse-transcribed polymerase
chain reaction (RT-PCR; TaqMan MicroRNAAssays; Applied Biosystems).
Quantitative RT-PCR mRNA analysis was performed in triplicate using the
SYBR Green master mix (Applied Biosystems) and the Bio-Rad iCycler
and normalized to GAPDH. Primers were from PrimerBank25 (primer
identification nos. 4885497a2, MYB; 4502735a3, CDK4; 4502741a2,
CDK6; and 7669492a3, GAPDH).
RACE PCR analysis
RACE PCR was performed using total RNA extracted from TPA-treated
K562 cells and the FirstChoice RLM-RACE Kit (Ambion) according to the
manufacturer’s instructions using primers: P1, 5⬘-AGAGCTTCCGAAGTCCTGG-3⬘ and P2, 5⬘-TTGCTCACAACAACCAGCTAAGA-3⬘ for the
5⬘-RACE PCR; and P3, 5⬘-ACCGGCCAGCTGTGAGTGTTTCTTT-3⬘
and P4, 5⬘-TGGCAGTGTCTTAGCTGGTTGTT-3⬘ for the 3⬘-RACE PCR.
Reaction products were analyzed by agarose gel electrophoresis, purified
using the High Pure PCR Cleanup Micro Kit (Roche Applied Science),
cloned (CloneJET PCR Cloning Kit), and sequenced.
Additional methods are provided in the supplemental data (available on
the Blood website; see the Supplemental Materials link at the top of the
online article).
Results
miRNA signature of TPA-induced MK differentiated K562 cells
To explore the role of miRNAs during K562 cell MK differentiation, we compared miRNA expression in undifferentiated versus
TPA-treated cells by miRNA microarray. Within 2 days of TPA
treatment, K562 cells stopped proliferating, became adherent, and
expressed the MK marker CD41 (supplemental Figure 1A, data not
shown). Total RNA was extracted from untreated K562 cells or
from cells cultured for 2 or 4 days after TPA treatment. Small RNA
libraries were prepared from each sample by size fractionation,
adapter ligation, and PCR amplification.23 cRNA was hybridized to
microarrays containing probes for 286 annotated miRNAs. miRNA
expression of samples harvested 2 and 4 days after TPA treatment
was similar (supplemental Figure 1B). Total miRNA expression
increased with differentiation (supplemental Table 1). We focused
on a subset of 12 highly up-regulated miRNAs, which included
miRNAs that increased by 6-fold or more and whose signal
saturated at day 4 (miR-34a, miR-375, miR-139, miR-409-3p, and
the miRNA clusters miR-132/212 and miR-221/222) and miRNAs
that increased by 50-fold or more (miR-181b, miR-299-5p, and
miR-134). We also selected miR-181a, which increased 19-fold,
because it is part of the miR-181 cluster (Figure 1A). The
up-regulation of each of these miRNAs was validated by Northern
blot (Figure 1B). One miRNA that was unchanged (miR-25) and
one that was down-regulated (miR-218) after TPA by microarray
analysis were probed as controls.
K562 cells can be differentiated into the erythroid lineage by
hemin.26 To determine whether the subset of highly up-regulated
miRNAs in TPA-treated K562 cells was specific to MK differentiation, we analyzed their expression in hemin-differentiated K562
cells by Northern blot. The 12 highly up-regulated miRNAs in
TPA-treated K562 cells were specific to MK because none was
up-regulated during hemin-induced erythroid differentiation (Figure 1C, supplemental Figure 1C). Furthermore, analysis of global
miRNA expression in TPA versus hemin-differentiated K562 cells
by miRNA microarray revealed very limited shared changes in
miRNA expression between both lineages.27 Of note, up-regulation
of the miR-24 clusters (containing miR-24, miR-23, and miR-27,
although up-regulation was ⬍ 6-fold) and down-regulation of
miR-17-5p/18a/20a and miR-106a/19b/92 clusters were common
to both differentiation pathways, suggesting that these miRNAs
might regulate common processes during terminal differentiation
of hematopoietic cells. miR-24 up-regulation during MK and
erythroid differentiation of K562 cells was also validated by
Northern blot (Figure 1C).
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
miR-34a REGULATES MEGAKARYOPOIESIS
2183
A
Counts
+ TPA
Fold
increase
miR-34a
644
65232
101
miR-181b
236
23255
99
miR-299-5p
277
19700
71
miR-134
167
8276
50
miR-375
1796
65231
36
miR-181a
1462
27359
19
miR-139
3619
65232
18
miR-222
3586
65232
18
miR-409-3p
4370
65231
15
miR-221
4722
65231
14
miR-212
6341
65232
10
miR-132
10695
65231
6
miR-299-5p
miR-132
miR-218
miR-212
miR-25
U6
U6
miR-34a inhibits proliferation, induces G1 arrest, and promotes
MK differentiation of K562 cells
Our next goal was to determine whether any of the miRNAs
up-regulated by TPA might play a role in regulating MK
differentiation. We first evaluated the effect of miRNA overexpression on MK markers. K562 cells were transfected with
miRNA mimics corresponding to each of the up-regulated
miRNAs or a control miRNA mimic and treated 48 hours later
with a suboptimal amount of TPA (0.1 nM), which does not
induce differentiation of untransfected or mimic controltransfected K562 cells (Figure 2A). MK differentiation was
assessed by flow cytometry for CD41 and CD61 (Figure 2A,
supplemental Figure 2A; data not shown). Overexpression of
miR-34a and miR-181a, but not the other up-regulated miRNAs,
induced expression of the MK integrins. Of note, CD41 was not
up-regulated without suboptimal TPA, even if cells were
transfected with miRNA combinations (data not shown).
We also evaluated the effect of miRNA overexpression on cell
proliferation. Overexpression of miR-34a and miR-134, but not
other miRNAs, inhibited cell proliferation in transfected K562 cells
treated with suboptimal TPA (0.1 nM, Figure 2B; data not shown).
To understand how these miRNAs might regulate cell proliferation,
we performed cell-cycle analysis (Figure 3). Both miRNAs per-
miR-375
miR-132
miR-409-3p
miR-212
miR-25
U6
U6
TPA
miR-24
miR-24
U6
U6
TPA day 4
day 3
Hemin
day 5
day 5
day 3
untreated
miR-181b
miR-299-5p
day 5
miR-181b
miR-34a
untreated
miR-375
miR-181a
day 3
miR-181a
miR-409-3p
miR-139
miR-222
Hemin
untreated
miR-134
miR-221
day 4
miR-222
day 2
miR-139
Hemin
untreated
miR-221
day 4
untreated
day 2
miR-34a
TPA
TPA day 4
C
TPA
day 4
untreated
B
Counts
- TPA
day 2
Figure 1. miRNA signature of TPA-treated K562 cells
differentiated into megakaryocytes. (A) miRNAs upregulated at least 6-fold 4 days after TPA treatment by
microarray. Background counts were ⱕ 500 and the
assay saturated at 65 232 counts. Reproducibility of the
miRNA microarray in 2 TPA-treated K562 samples is
shown in supplemental Figure 1B. (Supplemental Table 1
shows data for all miRNAs analyzed.) (B-C) Change in
miRNA expression after TPA or hemin treatment analyzed by Northern blot. U6 snRNA is a loading control.
The miRNAs most induced by TPA were not induced by
hemin (supplemental Figure 1C). MK differentiation by
TPA and erythroid differentiation by hemin were verified
(supplemental Figure 1A). miR-24 expression increased
after either TPA or hemin treatment.
turbed the cell cycle. miR-34a overexpression significantly increased the proportion of G1 phase cells and significantly
reduced the G2/M compartment, whereas miR-134 had the
opposite effect (reduced G1, increased G2/M). As expected,
when miRNA-transfected cells were treated with nocodazole for
16 hours, miR-34a–overexpressing samples showed increased
diploid cells (2N). However, no significant alteration of cellcycle profile was observed in cells transfected with 2 other
up-regulated miRNAs, miR-181a or miR-375. Taken together,
these results suggest that multiple miRNAs contribute to MK
differentiation of K562 cells and identify miR-34a as an
important contributor that regulates both cell-cycle progression
and MK marker expression.
miR-34a regulates MYB
Because miR-34a was the most up-regulated miRNA and exogenous miR-34a induced both MK differentiation markers and cellcycle arrest, we sought to identify miR-34a target genes in K562
cells. Candidate miR-34a target genes were chosen for experimental validation by combining in silico predictions28,29 and changes in
microarray mRNA expression after TPA treatment or enforced
expression of miR-34a (supplemental Tables 2-3). MYB, an important transcriptional regulator of hematopoietic cell differentiation,
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
NAVARRO et al
100
90
A
TPA 1 nM
101
102
103
104
100
101
102
103
104
control
0
0
untransfected
102
103
104
101
102
103
104
miRNA-181a
0
miRNA-34a
100
100
101
90
100
0
CELL NUMBER
120
100
120
0
0
NO TPA
Figure 2. miR-34a enhances megakaryocytic differentiation and inhibits proliferation in K562
cells. (A) Overexpression of miR-34a or miR-181a in
K562 cells induces the MK integrin CD41. K562 cells
were transfected with the indicated miRNA mimic
and 24 hours later stimulated with a suboptimal
amount of TPA (0.1 nM). Efficient uptake of dsRNA
oligonucleotides into transfected K562 cells is shown
in supplemental Figure 1D. CD41 expression was
analyzed by flow cytometry 72 hours after TPA stimulation. As positive control, K562 cells were treated
with 1 nM TPA. Levels of expression of the transfected miRNAs at the time of analysis are shown in
supplemental Figure 2B. (B) miR-34a and miR-134
inhibit K562 cell proliferation, assessed by the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) cell proliferation assay. Cells were transfected with the indicated miRNA and then treated
with a suboptimal amount of TPA as indicated.
Values are mean ⫾ SD.
101
102
103
104
100
101
102
103
104
90
100
miRNA-34a+181a
0
IgG1
CD41
100
101
102
103
104
FLUORESCENCE INTENSITY
Absorbance 570 nm
B
1.4
1.2
1.0
control
miR-34a
miR-221
miR-222
miR-134
0.8
0.6
0.4
0.2
0
0
12
24
48
Hours post TPA treatment
was an attractive candidate target gene. MYB mRNA and protein
were significantly down-regulated after TPA treatment of K562
cells by mRNA microarray (4.6-fold decrease; supplemental
Table 2), Northern blot, and immunoblot (supplemental Figure
6). After miR-34a overexpression in K562 cells, MYB mRNA
also decreased 1.9-fold by microarray analysis (supplemental
Table 3). To determine whether MYB is directly regulated by
miR-34a, the full-length MYB 3⬘-untranslated region (3⬘-UTR)
was cloned into a luciferase reporter plasmid, and the effect of
miR-34a overexpression on luciferase activity was assessed
(Figure 4A left). miR-34a overexpression reduced luciferase
activity by 45%. Overexpression of miR-25, which is not
predicted to regulate MYB and whose expression does not
change during TPA-induced differentiation, had no effect. The
3⬘-UTR of MYB has 3 rna22-predicted miRNA response element
(MREs; Figure 4A, supplemental Figure 3A). MRE2 is also
predicted by TargetScan as a high probability, although poorly
conserved, MRE with a 7-mer seed match. When each of the
predicted MREs was tested in luciferase assays, miR-34a only
regulated the reporter encoding MRE2 (Figure 4A right). The
70% reduction in luciferase expression from the MRE2containing reporter was almost as great as that obtained with a perfectly
complementary miR-34a sequence (90% reduction). Deletion of MRE2
or mutation of its seed in the context of the full-length 3⬘-UTR
significantly increased reporter luciferase activity (supplemental Figure
3B) but did not restore it to control levels. This suggests that, although
MRE2 is a bona fide binding site for miR-34a, other regions might
contribute to miR-34a regulation of MYB.
miR-34a overexpression in undifferentiated K562 cells reduced
MYB protein by 74% and mRNA by 40% (Figure 4B). Two other
miRNAs up-regulated after TPA treatment, miR-375 and miR181a, not predicted to target MYB, had no effect. MYB knockdown
using a retroviral vector encoding a MYB shRNA also significantly
up-regulated CD41 expression in untreated K562 cells, which was
further increased by adding a suboptimal amount of TPA (0.1 nM;
Figure 4C). The increased CD41 expression after MYB knockdown
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
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untreated
miR-375
control
G1: 31
S: 44
G2/M: 25
600
400
G1: 31
S: 42
G2/M: 27
600
400
miR-134
miR-34a
G1: 41
S: 37
G2/M: 22
600
400
400
200
200
200
0
0
0
0
0
200 400 600 800 1000
0
200 400 600 800 1000
0
200 400 600 800 1000
miR-181a
G1: 24
S: 44
G2/M: 32
600
200
CELL NUMBER
Figure 3. miR-34a and miR-134 inhibit
cell-cycle progression at different phases
of the cell cycle. K562 cells, transfected
with the indicated miRNA mimic, were analyzed by propidium iodide staining 48 hours
later. Replicate samples were either left
untreated or treated with nocodazole
(100 ng/mL) for 16 hours before propidium
iodide staining. The percentage of cells in
the G1, S, or G2 phase of the cell cycle is
indicated. The bar graph at the bottom
represents the mean ⫾ SD of 4 independent
experiments. #Significant differences
(P ⬍ .05) relative to the control sample by
Student 2-sided t test.
miR-34a REGULATES MEGAKARYOPOIESIS
G1: 30
S: 44
G2/M: 26
600
400
200
0
0
200 400 600 800 1000
0
200 400 600 800 1000
+ nocodazole
control
miR-375
600
miR-34a
600
4N
4N
0
0
0
200 400 600 800 1000
0
400
400
2N
200
2N
200
2N
200
0
200 400 600 800 1000 0
4N
600
4N
400
200
2N
miR-181a
600
800
4N
400
400
200
miR-134
600
200 400 600 800 1000
2N
0
0
0
200 400 600 800 1000
0
200 400 600 800 1000
FLUORESCENCE INTENSITY
60
G1
S
G2/M
#
% of total cells
50
40
#
#
30
#
20
10
was comparable with that obtained when miR-34a was overexpressed (Figure 2A). Analogous results were obtained after transfection with 3 different MYB siRNAs (not shown). Cell proliferation
also decreased in MYB knockdown samples (not shown), consistent
with previous observations.30
The TargetScan algorithm predicts 5 of the other highly
up-regulated miRNAs (miR-132/212, miR-221/222, and miR-134)
as potential MYB regulators. We therefore analyzed MYB protein
in K562 cells overexpressing these miRNAs and tested by luciferase reporter assay their regulation of the full-length MYB 3⬘-UTR
(supplemental Figure 4). miR-221 and miR-134 also directly
regulated MYB, although not as strongly as miR-34a. Therefore,
miR-34a and other up-regulated miRNAs suppress MYB expression.
miR-34a down-regulates CDK4 and CDK6 in K562 cells
CDK4 and CDK6 have previously been shown to be regulated by
miR-34a in colon, lung, and prostate tumor cell lines.17,19,31,32
Because these kinases drive progression from G1 to S phase and
overexpressing miR-34a increases the G1 compartment (Figure 3),
it is probable that these kinases are also regulated by miR-34a in
K562 cells. Both CDK4 and CDK6 mRNAs are also downregulated in K562 cells after TPA treatment (3.7- and 2.6-fold,
respectively; supplemental Table 2) or enforced miR-34a expression (3.2- and 3.0-fold, respectively; supplemental Table 3).
1a
-18
miR
4
-13
miR
a
-34
miR
5
-37
miR
con
trol
0
Furthermore, CDK4 and CDK6 proteins decreased in TPAtreated cells by immunoblot (supplemental Figure 6). Previous
reports identified one CDK4 and 2 CDK6 miR-34a MREs.17,19,32
To verify that miR-34a regulates CDK4 and CDK6 in K562
cells, we analyzed their expression by immunoblot. Overexpressing miR-34a significantly reduced CDK4 and CDK6 protein
(89% and 87%, respectively; Figure 5A). Overexpression of
miR-299-5p, which is not predicted to target either gene, did not
alter either. In addition, miR-34a overexpression in K562 cells
also significantly reduced CDK4 and CDK6 mRNAs analyzed
by quantitative PCR (Figure 5A). rna22 predicts 8 CDK6
miR-34a MREs, of which MRE3 is also predicted by TargetScan
(supplemental Figure 5A). Because these MREs have not been
previously tested, we analyzed their activity in luciferase
reporter assays (supplemental Figure 5B). Insertion of CDK6
MRE3 and MRE5 strongly reduced luciferase activity (80% and
65%, respectively), whereas MRE2 and MRE8 each had a
moderate effect (25% and 40%, respectively).
Knockdown of both CDK4 and CDK6 significantly reduced
cell proliferation by causing cells to accumulate in G1, but
knocking down either by itself had no significant effect on
proliferation (Figure 5B). CDK6 knockdown on its own slightly,
but significantly, increased the proportion of G1 phase cells
(38% vs 28% in control cells). These experiments are consistent
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A
MYB mRNA
MRE 34a (3)
MRE 34a (2)
MRE 34a (1)
TAG
554 666
532
688
1070
1092
AAAAAAA
MRE, miRNA response element
polyA
SV40
promoter
MRE
100
80
60
40
20
0
100
60
40
20
NO
0
1
0.8
0.6
0.4
0.2
shRNA
vector
miR
-34
a
-18
1a
0
miR
1.14 0.26 0.92
80
1.2
con
trol
Relative MYB gene expression
miR-375
α-tubulin
1
120
mi
R34
a
mi
R25
miR-34a
miR-181a
control
MYB
Relative ratio
140
MR
E
MR
EM
yb
(1
MR
)
EM
yb
(2
MR
)
EM
yb
(3
)
AS
-3
4a
% relative luciferase activity
120
co
ntr
ol
% relative luciferase activity
c-Myb 3’UTR
B
polyA
renilla luciferase
TPA 1nM
MYB
IgG1
CD41
α-tubulin
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Figure 4. miR-34a directly regulates MYB expression. (A) miR-34a regulates luciferase activity of a reporter vector containing either the full-length 3⬘-UTR of MYB (left) or
individual predicted MRE (right). Relative luciferase activity was assayed in 293T cells 48 hours after cotransfection with a luciferase reporter plasmid and the indicated miRNA
mimic. AS-34a designates a reverse complementary sequence of miR-34a. (B) Overexpression of miR-34a in K562 cells down-regulates MYB protein and mRNA. K562 cells,
transfected with the indicated miRNA mimic, were analyzed for MYB protein (left) and mRNA (right) by immunoblot or quantitative PCR, respectively, 48 hours later. Protein
expression relative to ␣-tubulin was quantified by densitometry. mRNA normalized to GAPDH mRNA is shown relative to the ratio in K562 cells transfected with a miRNA mimic
control. (C) MYB knockdown (right) up-regulates the MK-specific marker CD41 in cells treated with a suboptimal amount of TPA (0.1 nM). As positive control for CD41
induction, K562 cells were treated with 1 nM TPA.
with previous work showing that CDK4 and CDK6 have
redundant roles in controlling the G1 to S transition33,34 and
highlight the potential relevance of miR-34a as a regulator of
both kinases.
To address whether miR-34a down-regulates MYB, CDK4, and
CDK6 during MK differentiation, we analyzed the change of
expression of miR-34a and its targets over time in TPAdifferentiated K562 cells (supplemental Figure 6). miR-34 became
faintly detectable 12 hours after adding TPA and increased over the
next 4 days. However, MYB and CDK4 were down-regulated
before miR-34a was up-regulated. CDK6 expression correlated
inversely with the induction of miR-34a. Of note, MYB was
transiently reexpressed 24 to 48 hours after TPA treatment and
became undetectable again only when miR-34a was highly expressed. Similarly, CDK4 protein levels increased at later time
points. These data suggest that, although miR-34a might not be
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
CDK4
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Figure 5. miR-34a regulates CDK4 and CDK6 in K562 cells. (A) K562 cells, transfected with the indicated miRNA mimic, were analyzed for CDK4 and CDK6 protein and mRNA by
immunoblot (left) or quantitative PCR (right), respectively, 48 hours later. Protein expression relative to ␣-tubulin was quantified by densitometry. mRNA normalized to GAPDH mRNA is
shown relative to the ratio in K562 cells transfected with a miRNA mimic control. (B) Simultaneous knockdown (top left) of CDK4 and CDK6, but not either alone, inhibits proliferation and
increases the G1 compartment in K562 cells. Cell proliferation was evaluated using the MTT cell proliferation assay (top middle). Cell-cycle analysis (representative flow cytometry
histograms, bottom) was performed 72 hours after transfection in nocodazole-treated and untreated cells. The bar graph (top right) represents the mean ⫾ SD of 4 independent
experiments performed without nocodazole synchronization. Statistical analysis was performed using the Student 2-sided t test: #P ⬍ .05; **P ⬍ .01.
responsible for the initial down-regulation of CDK4 or MYB,
miR-34a might act to suppress their reexpression to maintain the
postmitotic MK state.
Enforced miR-34a expression enhances thrombopoietin-induced
MK differentiation of primary human CD34ⴙ cells
Because results in hematopoietic cell lines may not reflect normal
hematopoiesis, our next goal was to determine whether miR-34a also
contributes to MK differentiation of primary human hematopoietic stem
cells (HSCs) treated with TPO, IL-3, and SCF.35 MK differentiation was
assessed by flow cytometry for CD41 and CD61 staining (Figure 6A).
CD41 and CD61 were detected by day 6 and increased over 2 weeks,
when most cells expressed these markers. miR-34a expression analyzed
by quantitative PCR increased with MK differentiation, first becoming
significantly increased at day 10 (Figure 6B). MYB, CDK4, and CDK6
mRNA also decreased with MK differentiation. As with K562 cells, the
kinetics of miR-34a induction and down-regulation of its targets did not
correlate. In particular, MYB and CDK6 were down-regulated before
miR-34 expression became appreciable. These findings suggest that
miR-34-independent mechanisms control the initial down-regulation of
these genes.
To determine whether miR-34a contributes to MK differentiation of HSCs, CD34⫹ cells infected with lentiviruses encoding
miR-34a or a control sequence were evaluated in CFU-MK
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
NAVARRO et al
120
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B
#
#
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control miR-34a miR-150 miR-30c
Figure 6. Enforced miR-34a expression enhances MK differentiation of human CD34ⴙ hematopoietic precursors. (A) CD34⫹ hematopoietic cells, differentiated to MK
by culture in medium containing TPO, SCF, and IL-3, express MK integrins. The proportion of CD41/CD61-positive cells is indicated. (B) Changes in miR-34a and its MYB,
CDK4, and CDK6 target mRNAs were evaluated by quantitative PCR. miR-34a expression is normalized to U6 and mRNA is normalized to GAPDH. Gene expression is plotted
as expression relative to day 0. ND indicates not detected. (C) CFU-MK assay of miRNA-overexpressing CD34⫹ HSCs. Primary human CD34⫹ cord blood cells were infected
with lentiviruses encoding the indicated miRNAs, and the effect of miRNA overexpression on CFU-MK numbers was evaluated 12 days later. The bar graph represents the
mean ⫾ SD of quadruplicate samples. A representative experiment of 3 independent experiments is shown. #P ⬍ .05 relative to control lentivirus-infected cells. Exogenous
miR-34a and miR-150 both enhance MK colony numbers.
assays (Figure 6C). miR-150, a known regulator of the MK
lineage,7 and miR-30c, whose expression did not change in
TPA-induced megakaryopoiesis (supplemental Table 1), were
used as additional controls. miR-34a significantly increased MK
colony number by approximately 30%, whereas the miR-30c–
expressing virus had no significant effect. As expected, miR-150
also increased MK colonies (⬃ 60%). Therefore, miR-34a is
able to enhance MK differentiation of primary HSCs, although
to a lesser extent than miR-150.
miR-34a up-regulation in TPA-differentiated K562 cells is
p53-independent
miR-34a participates in the p53 gene regulatory network.16 However, K562 cells are p53-null.36 K562 cells contain a single cytosine
insertion in exon 5, between codons 135 and 136, of TP53, which
generates a frameshift mutation leading to an N-terminal truncated
protein of 147 amino acids. Although a shorter transcript can be
detected by RT-PCR, neither the truncated nor full-length protein is
detected by immunoblot (Figure 7B).36 miR-34a is contained
within the second exon of an expressed sequence tag (accession no.
DB286351) encoded by the negative strand of human chromosome
1.15,17,18 Exon 1 is separated from exon 2 by a 30-kb intron and
contains a conserved p53 binding site responsible for miR-34a
transactivation. The p53-regulated pri-miR-34a promoter region
has been identified 1.5 kb upstream and 0.5 kb downstream of the
pri-miR-34a transcription start site (TSS), which includes the
conserved p53 binding site.15,17,18 To evaluate whether the identified promoter region drives expression of pri-miR-34a during
TPA-induced differentiation of K562 cells, we cloned the putative
promoter of expressed sequence tag DB286351 into the promoterless
luciferase reporter vector pGL3-basic. Luciferase activity driven
by the putative miR-34a promoter was not enhanced by TPA
treatment in K562 cells (Figure 7A). TPA treatment increased
luciferase activity approximately 400-fold from a control reporter
gene in which the luciferase gene was driven by the TPAresponsive CD41 promoter. The putative miR-34a promoter reporter vector was strongly activated (100-fold relative to the
promoterless reporter) when a p53 expression plasmid was cotransfected (Figure 7B). These results confirm that K562 cells are
p53-null and demonstrate that miR-34a up-regulation during
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
B
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pGL3+p53
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miR-34a REGULATES MEGAKARYOPOIESIS
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E2
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Chr 1
E1’ 18.8 Kb E2’
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pGL3-Basic PM-34a PM-34a-K1/2
124 bp
1.3 Kb
E1
291 bp
30.1 Kb
pre-miR-34a
E2
200 bp
pri-miR-34a-K2
671 bp
Figure 7. miR-34a up-regulation after TPA treatment of K562 cells is p53-independent. (A) The p53-dependent miR-34a promoter does not activate luciferase expression
after TPA treatment of K562 cells. Cells were transfected with a firefly luciferase reporter vector in which luciferase expression was driven by the DB286351 miR-34a promoter
(PM-34a) or the CD41 promoter (PM-CD41) or with the promoterless luciferase vector (pGL3-Basic). Samples were cotransfected with a Renilla luciferase reporter vector for
normalization. Luciferase activity was measured 48 hours after TPA treatment. (B) Exogenous expression of p53 in K562 cells activates the DB286351 miR-34a promoter.
K562 cells were transfected with promoterless firefly luciferase reporter vector (pGL3) or a reporter containing the miR-34a promoter (PM-34a), a Renilla luciferase reporter
vector, and increasing amounts (2, 4, and 8 ␮g) of pCMV-p53 vector. The total amount of transfected DNA was kept constant using an empty pCMV plasmid. Luciferase activity
was measured 48 hours after transfection and was normalized as described. The fold increase in luciferase activity relative to the control sample transfected with the
promoterless vector is shown. The bottom panel represents the level of p53 protein in the samples analyzed herein by immunoblot. The membrane was stripped and reprobed
for ␣-tubulin as a loading control. (C) 5⬘- and 3⬘-end RACE PCR analysis of pri-miR-34a transcripts in TPA-treated K562 cells. Agarose gel images show the 5⬘- and 3⬘-RACE
PCR products obtained in K562 cells treated with TPA for 4 days. (D) Two pri-miR-34a transcripts identified by sequencing the 5⬘-end RACE PCR products from TPA-treated
K562 cells and their location in the genome. E indicates exon; the numbers below the exons indicate their length in base pairs. Also indicated is the length of the intronic regions.
For comparison, the previously described pri-miR-34a transcript, DB286351, is also shown (top). (E) The genomic region adjacent to the alternate TSS serves as the miR-34a
promoter in TPA-treated K562 cells. A DNA genomic fragment comprising 1.5 kb upstream to 0.5 kb downstream of the identified alternative TSS (PM-34a-K1/2) was cloned
into pGL3-basic, and the effect of TPA treatment on luciferase activity was evaluated as described.
TPA-induced K562 cell differentiation is p53-independent. Therefore, DNA regulatory sequences other than those in the previously
described miR-34a promoter drive miR-34a expression.
To identify the promoter of the pri-miR-34a transcript in K562
cells, we performed 5⬘-RACE PCR analysis using primers that
annealed just downstream of the miR-34a sequence (Figure 7C,
supplemental Figure 7A). The 5⬘-RACE PCR product revealed an
alternate TSS approximately 20 kb upstream of the previously
described TSS (Figure 7D, supplemental Figure 7B). Two different
pri-miR-34a transcripts were sequenced that contained 2 exons in
addition to exon 1 and exon 2 in pri-miR-34a DB286351 (Figure
7D, supplemental Figure 8). These pri-miR-34 transcripts shared
the same TSS and exon E1⬘ but differed in exon E2⬘. Of 14 clones
sequenced, 13 corresponded to pri-miR-34a-K1, whereas only
1 corresponded to pri-miR-34a-K2, suggesting that pri-miR-34a-K1
was the dominant transcript in TPA-treated K562 cells. To evaluate
whether regions adjacent to the TSS could be responsible for
TPA-induced up-regulation of miR-34a, a 2-kb genomic DNA
fragment (1.5 kb upstream to 0.5 kb downstream of the alternate
TSS) was cloned into pGL3-basic. TPA treatment increased
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2190
NAVARRO et al
luciferase activity approximately 40-fold, whereas no significant
increase was observed for the construct driven by the previously
described miR-34a promoter (Figure 7E). Therefore, a p53independent TPA-inducible promoter regulates expression of distinct pri-miR-34a transcripts in p53-null K562 cells. Further work
is required to define the transcription factors that regulate the
p53-independent phorbol ester–responsive transcripts.
Discussion
Twelve miRNAs (miR-34a, miR-134, miR-139, miR-299-5p, miR375, miR-409-3p and the miRNA clusters miR-132/212, miR-181a/
181b, and miR-221/222) were strongly up-regulated (6- to 100fold) in TPA-differentiated K562 cells. This miRNA signature was
specific to MK differentiation because none of these miRNAs was
up-regulated in hemin-differentiated cells. Our results are consistent with a report that down-regulation of miR-221/222, which
derepresses c-kit expression, is required for erythroid differentiation of CD34⫹ hematopoietic precursors.37 Therefore, we can
speculate that up-regulation of miR-221/222, and perhaps other
miRNAs, might be required to suppress genetic programs that
otherwise would favor erythroid differentiation.
To gain insight into the functional relevance of the miRNAs
up-regulated during MK differentiation, we analyzed the effect of
enforced expression of these miRNAs on MK differentiation of
K562 cells. None of the miRNAs alone or in combination induced
MK differentiation unless the cells were also exposed to suboptimal TPA. This suggests that either the up-regulated miRNAs on
their own cannot regulate differentiation or that comanipulation of
other miRNAs that were less profoundly modulated (in either
direction) might be required. However, when cells were treated
with suboptimal TPA concentrations that did not induce MK
differentiation on their own, both miR-34a and miR-181a induced
the MK marker CD41. In addition, miR-34a and miR-134 inhibited
cell proliferation, altering the cell cycle at different points. miR-34a
overexpression induced a G1 phase arrest, whereas miR-134 caused
an arrest in G2/S. miR-34a was not only the most up-regulated
miRNA during K562 cell MK differentiation, but enforced expression of miR-34a facilitated 2 important features of MK differentiation: cell-cycle arrest and induction of MK marker genes. Therefore, our results suggest that miR-34a might play an especially
important role in MK differentiation.
miR-34a directly inhibits expression of the transcription factor
MYB, an important regulator of cell differentiation at multiple
decision forks in hematopoiesis.38 Previous studies in mice provided compelling evidence supporting a role for MYB as a potent
negative regulator of megakaryopoiesis. Mice bearing mutations
that compromised MYB function had elevated blood platelet and
bone marrow MK counts.38 In addition, evidence supporting a
crucial role for MYB as a regulator of the E-MK lineage
bifurcation was provided by 2 studies showing that reduced MYB
expression in MK/erythrocyte lineage–restricted progenitors increased MKs and reduced erythroid progenitor cell numbers.39,40
In addition to regulation by miR-34a as demonstrated here,
MYB has also been shown to be regulated by miR-15a during
erythroid differentiation of human CD34⫹ cells and by miR-150
both in B-cell development and to control MEP differentiation in
mice.7,41 However, it is doubtful that these 2 miRNAs regulate
MYB expression during MK differentiation of K562 cells because
miR-15a is not significantly changed during TPA-induced MK
differentiation of K562 cells by microarray (supplemental Table 1),
BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
and miR-150, whose signal increases in TPA-treated K562 cells by
microarray, could not be detected by Northern blot in either
untreated or TPA-treated K562 cells (supplemental Figure 1E). The
MYB 3⬘-UTR encodes for evolutionarily conserved, high likelihood TargetScan-predicted MREs capable of being regulated by
miR-150 (3 conserved, 1 nonconserved site) and miR-15/16
(2 conserved sites) as well as for the hematopoietic cell-expressed
regulator miR-155 (2 conserved sites) and for miR-200 (1 conserved, 1 nonconserved site). Of note, the miR-34a MRE in the
3⬘-UTR of MYB is not conserved in mammals. Five other strongly
up-regulated miRNAs in our system (miR-134, miR-221/222,
miR-132/212) also have nonconserved MYB MREs.28 Of these,
miR-221 and miR-134 also appear to regulate MYB in K562 cells.
Because down-regulation of MYB is critical for hematopoietic cell
differentiation, it is probable that MYB expression is regulated by
multiple miRNAs in different hematopoietic contexts. In addition,
miRNA-independent mechanisms to down-regulate MYB, including transcriptional regulation, RNA splicing, and proteolytic degradation via ubiquitylation, are also important.42-44 In particular, our
kinetics data suggest that miR-34a–independent regulation of MYB
expression is needed for initially suppressing MYB expression.
miR-34a is not substantially up-regulated until a day after TPA
stimulation of K562 cells. This finding suggests that miR-34a does
not participate in the earliest differentiating events but acts to
suppress expression of its target genes at a later stage, perhaps to
preserve the postmitotic block and maintain the MK lineage.
Consistent with this hypothesis is the temporal association of
miR-34a expression with late suppression of reinitiated MYB and
CDK4 expression. Careful kinetic studies will be required to sort
out whether other MYB-regulating miRNAs, such as miR-150,
miR-221, or miR-134, might play a role in the initial downregulation of these targets. Importantly, exogenous expression of
miR-34a in primary CD34⫹ HSCs enhanced MK colony formation
in a more physiologic setting. However, further studies using
normal hematopoietic precursor cells are needed to determine
whether miR-34a function is mediated by regulation of additional
targets other than the ones identified in the present study.
The postulated role of miR-34a as a tumor suppressor is
supported by our findings that enforced expression of miR-34a
arrests K562 cells in the G1 phase of the cell cycle. In this study, we
confirmed that miR-34a suppresses the expression of the CDKs
that control the G1 to S transition (CDK4 and CDK6) and identified
additional miR-34a MREs in the 3⬘-UTR of CDK6. Knockdown of
CDK4 and CDK6, but not of either by itself, mimicked the cellcycle inhibitory effect of overexpressing miR-34a, suggesting that
these CDKs are important miR-34a target genes.
Down-regulation of miRNAs could also be important for
megakaryopoiesis. One study suggested that down-regulation of
miR-146a via transcriptional inhibition by PLZF and consequent
derepression of the miR-146a target gene CXCR4 facilitates
megakaryopoiesis.45 Another suggested that miR-155 downregulation during megakaryopoiesis facilitates MK differentiation
by derepressing expression of Ets-1 and Meis1.46 However, these
miRNAs were not significantly altered by microarray after TPA
treatment of K562 cells. Our seemingly discrepant findings could
be the result of differences in timing or to idiosyncratic properties
of the cell line, which might not reflect normal cell differentiation.
Several groups have shown that p53 transcriptionally activates miR-34a, which in turn regulates the expression of
p53-regulated genes.16 However, K562 cells are p53-null.36
Therefore, miR-34a expression is not driven by p53 in these
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BLOOD, 3 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 10
cells. In this study, we identified alternate pri-miR-34a transcripts induced by TPA. Furthermore, the genomic region
adjacent to the alternate TSS used by these transcripts drives
their TPA-dependent expression. The tumor suppressor function
of miR-34a could therefore be induced by p53 and/or by a
phorbol ester-responsive promoter in p53-null tumor cells.
miR-34a may serve to modulate the tumor-promoting effects of
phorbol esters. The biologic effect of phorbol esters is mediated
largely by protein kinase C, which activates key transcription
factors, especially AP-1 (fos/jun) and nuclear factor-␬B.47-49
PKC-activated transcription factors, such as these, probably
bind to the promoter region surrounding the TSS of the
TPA-dependent pri-miR-34a transcripts. It will be interesting to
analyze whether miR-34a can be expressed in a p53-independent manner in p53-sufficient or p53-null tumor cells of
nonhematologic origin and in normal cells. Activation of
miR-34a by itself may offer a novel useful approach to tumor
therapy by interfering with cell proliferation.
Note added in proof: While this manuscript was in press, Kato
et al reported that miR-34 induction postirradiation in Caenorhabditis elegans does not require the p53 homolog cep-1.50
miR-34a REGULATES MEGAKARYOPOIESIS
2191
Acknowledgments
The authors thank Gary Gilliland, Oliver Hoffman, and the
laboratory scientists (in the laboratory of J.L.) for useful discussions.
Authorship
Contribution: F.N. designed the research, performed the experiments, analyzed the data, and wrote the manuscript; D.G. performed the experiments; E.M. performed the miRNA microarray
analysis; M.C. assisted in gene microarray data analysis; I.R.
assisted in miRNA target identification; Z.B. performed the miRNA
microarray analysis; and J.L. designed the research, analyzed the
data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Judy Lieberman, Immune Disease Institute, Harvard Medical School, Warren Alpert Bldg Rm 255, 200 Longwood Ave,
Boston, MA 02115; e-mail: [email protected].
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Most mammalian mRNAs are conserved targets
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2. Cobb BS, Nesterova TB, Thompson E, et al. T
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