1. No category

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reports
Down-regulation of the cyclin E1 oncogene expression by
microRNA-16-1 induces cell cycle arrest in human cancer cells
1
2
1
1,
Fu Wang , Xiang-Dong Fu , Yu Zhou & Yi Zhang *
1
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P. R. China, 2Department of
Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651
Cyclin E1 (CCNE1), a positive regulator of the cell cycle,
controls the transition of cells from G1 to S phase. In numerous
human tumors, however, CCNE1 expression is frequently
dysregulated, while the mechanism leading to its dysregulation
remains incompletely defined. Herein, we showed that CCNE1
expression was subject to post-transcriptional regulation by a
microRNA miR-16-1. This was evident at protein level of CCNE1
as well as its mRNA level. Further evident by dual luciferase
reporter assay revealed that two evolutionary conserved binding
sites on 3’ UTR of CCNE1 were the direct functional target sites.
Moreover, we showed that miR-16-1 induced G0/G1 cell cycle
arrest by targeting CCNE1 and siRNA against CCNE1 partially
phenocopied miR-16-1-induced cell cycle phenotype whereas
substantially rescued anti-miR-16-1- induced phenotype. Together,
all these results demonstrate that miR-16-1 plays a vital role in
modulating cellular process in human cancers and indicate the
therapeutic potential of miR-16-1 in cancer therapy. [BMB reports
2009; 42(11): 725-730]
INTRODUCTION
Progression of a cell through or its arrest in a specific cell cycle
phase requires the integration, processing and initiation of a
variety of signal transduction pathways. Critical for the function of the cell cycle machinery are the activities of cyclin
dependent kinases (Cdks) and their activating subunits, the
cyclins. Cyclin E1 (CCNE1), an essential cyclin activating Cdk2,
regulates the G1-S phase transition of the mammalian cell
division cycle (1). Its timing expression plays a direct role in
the initiation of DNA replication (2), the control of histone
biosynthesis (3), and the centrosome cycle (4).
CCNE1 expression is largely restricted to the G1-S phase
transition in normal dividing cells. Previous studies have revealed that such a periodical expression of CCNE1 is controlled by transcriptional regulation and ubiquitin-dependent
*Corresponding author. Tel: 86-27-68756207; Fax: 86-27-68754945;
E-mail: [email protected]
Received 4 March 2009, Accepted 25 March 2009
Keywords: Cancer therapy, CCNE1, Cell cycle arrest, miR-16-1,
Post-transcriptional regulation
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proteolysis (5). However, in different types of human cancers,
CCNE1 expression is uncoupled from cell cycle progression
(6, 7). A growing studies suggest that CCNE1 is expressed
significantly higher than physiological levels in many human
tumors, in particular breast cancer, and also non-small cell
lung cancer, leukemia and others (8). And the genomic locus
at which the CCNE1 gene is located (19q12-q13) is frequently
amplified in human cancers (9, 10). Transgenic mouse models
where CCNE1 is constitutively expressed develop malignant
diseases (11), which further supporting the notion of CCNE1 as
a dominant oncogene. Clearly, uncoupling CCNE1 expression
from the cell cycle control is a critical factor in human cancer
development, while the known regulatory mechanisms cannot
fully explain such a dysregulation.
MicroRNAs (miRNAs) have recently come into focus as a
novel class of post-transcriptional regulatory elements. They
are abundant endogenous, 22-nucleotides (nt) RNAs and function by mainly binding to complementary sites on 3’ untranslated region (3’ UTR) of multiple target mRNAs to repress the
target translation or induce the target degradation (12). A
number of miRNAs have been found to be involved in a large
variety of cellular processes, including cell proliferation, differentiation, cell cycle and apoptosis (13)，and their aberrant
expression has been linked to disease. Emerging studies have
uncovered both the tumor suppressive and oncogenic potential of a number of miRNAs (14, 15), underscoring their importance in human cancers.
The findings of the generally altered miRNA expression in
different tumors led us to question if CCNE1 mRNA is
normally under the regulation of microRNAs. We initially
conducted a survey of the potential microRNAs that can bind
to CCNE1 mRNA using bioinformatics databases. MiR-16-1
was identified as a strong candidate due to its crucial function
in cell cycle control (16-18). We undertook studies to confirm
the predicted role of miR-16-1 in the regulation of CCNE1.
Additionally, we showed that miR-16-1 induced a significant
G0/G1 cell cycle arrest, which at least in part through downregulation of CCNE1.
RESULTS
Two evolutionary conserved target sequences for miR-16-1
are found in the 3’ UTR of CCNE1
Several prediction programs have been developed to identify
potential miRNA targets (19-21). In initial studies, we adopted
the bioinformatic approach to identify miRNAs with putative
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microRNA-16-1 downregulates cyclin E1 expression
Fu Wang, et al.
Fig. 1. Two conserved miR-16-1 target
regions in the 3’ UTR of CCNE1. (a)
Diagram showing the predicted results
from database miRGator. (b) Schematic
represention of the two putative miR16-1 binding sites in the 3’ UTR of
CCNE1. (c) Comparison of nucleotides
between the miR-16-1 seed sequence
and its two target sites in five species.
binding sites in CCNE1 3’ UTR. By the on-line search of
miRGator (22), an integrated database of three most used
software algorithms (TargetScan, PicTar, and MiRanda), we
found that 3 candidate miRNAs were in the intersection of
three programs (Fig. 1a). Of the 3 miRNAs, 2 miRNAs (miR16-1, and miR-195) share the identical seed region and belong
to the miR-16 family. The other microRNA is miR-138, whose
function is poorly understood. As noted, miR-16-1 is considered
to be an important regulator of cell cycle (16-18) and has been
found downregulated in many cancers (23, 24). Therefore, in
this study, we have focused on studying the role of miR-16-1
in regulating CCNE1 expression.
As shown in Fig. 1b, there are two potential target sites in 3’
UTR of CCNE1 for miR-16-1, and the calculated minimum free
energies for its hybridization with the predicted target sites are
∼-24 kcal/mol and ∼-24.8 kcal/mol, determined by RNA
hybrid analysis (25), which is consistent with the authentic
miRNA targeting (26). Comparing the human sequence for
interspecies homology, we found that the miR-16-1 target
sequences at nt229∼254 and nt459-492 of the CCNE1 3’
UTR are highly conserved among five species (Fig. 1c).
Fig. 2. miR-16-1 regulates CCNE1 expression at the post- transcriptional level. (a) MCF-7 cells were transfected a miR-16-1 overexpression plamid or a control plasmid. 72 hours later, GFP- positive
cells were sorted by flow cytometry. Then the cell lysates were used
to detect CCNE1 protein by western blot. Analysis of β-actin was performed as a loading control. (b) qRT-PCR of the mRNA levels in
MCF-7 cells transfected with the miR-16-1 overexpressed plasmid or
control plasmid. The mRNA levels of the CCNE1 was shown and normalized against that of GAPDH. The ctrl value was set to 1. Error
bars are means of three separated experiments.
miR-16-1 downregulates CCNE1 expression
To determine whether miR-16-1 downregulate the endogenous
CCNE1 expression, we first generated a construct that can
express precursor forms of miR-16-1 and GFP under the control
of a CMV promoter. The GFP gene was interrupted by the
genomic fragment encoding the endogenous miR-16-1 locus
and its correct splicing from GFP gene resulted in expression
of miR-16-1. The advantage of this construct is that we can
select miR-16-1 expression cells by sorting GFP-positive cells.
Then human breast cancer MCF-7 cells, in which CCNE1 is
overexpressed, were transfected with this miR-16-1 overexpression plasmid or a control plasmid. After sorting GFPpositive cells by flow cytometry, western blot analysis were
performed. The results showed that enforced expression of
miR-16-1 led to a significant decrease in endogenous CCNE1
proteins (Fig. 2a), suggesting that the endougenous expression
of CCNE1 is downregulated by miR-16-1.
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We further investigated whether miR-16-1 also targeted
CCNE1 mRNA for degradation by real-time PCR analysis of
RNAs isolated from MCF-7 cells transfected with the miR-16-1
overexpression plamid or control plasmid. As shown in Fig.
2b, CCNE1 mRNA levels in cells transfected with miR-16-1
overexpression plasmid were correspondingly decreased about
60% compared to that in cells transfected with the control
plasmid. Together, these observations demonstrate that miR16-1 downregulates the expression of CCNE1, which is at least
in part due to the induction of the target mRNA degradation.
CCNE1 is a direct target of miR-16-1
The predicted interaction between miR-16-1 and its target sites
in CCNE1 3’ UTR is illustrated in Fig. 3a. To determine whether the negative regulatory effects of miR-16-1 on CCNE1
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microRNA-16-1 downregulates cyclin E1 expression
Fu Wang, et al.
Fig. 3. CCNE1 is a direct target of miR-16-1.
(a) Diagram of the putative base-pairing between miR-16-1 and its wild-type or mutated
target sites in the 3’ UTR of CCNE1. Bold dots
indicate the G:U base-pairings, asterisks highlight the mutated nucleotides in CCNE1-MUT.
(b) Dual luciferase reporter assay. Renilla liciferese constructs pRL-CCNE-wt, mut1, mut2,
mut1&2 were respectively contransfected into
HEK293 cells with a firefly luciferase control
plasmid pGL3-promoter together with synthetic
miR-16-1 duplex or control duplex. Mut1&2
construct contains both of nucleotides mutations in constructs mut1 and mut2. Shown are
relative luciferase values normalized to tranfections with control duplex, whose value was
set to 1. Each bar represents the value from
three independent experiments. (c) Dual luciferase reporter assay as in (b) was performed
using 2’-O-Methylated anti-miR-16-1 inhibitor
or scramble control RNA oligos to transfect
HeLa cells.
expression were indeed mediated through binding to the
predicted target sites at the 3’ UTR of target mRNAs, we
cloned the full length 3’ UTR of CCNE1 immediately downstream of the renilla luciferase open reading frame (ORF) in
the plasmid pRL-TK. Transient transfection of HEK293 cells
with synthetic miR-16-1 duplex and the pRL-CCNE1-3’ UTR
reporter construct, led to a significant decrease of the luciferase reporter activity as compared with the control miRNA
duplex (Fig. 3b).
To further investigate which putative target site was regulated
by miR-16-1, we introduced point mutation to the corresponding seed sequences at pRL-CCNE1-3’ UTR to eliminate
the predicted binding by miR-16-1 (Fig. 3a). As shown in Fig.
3b, suppression of the reporter activity by miR-16-1 was
partially relieved by mutation of the single conserved seed
complementary site, and mutations of both seed complementary sites almost fully rescued the repression for CCNE1, denoting that both of the two matching sites identified strongly
contribute to the miRNA:mRNA interaction that mediates the
post-transcriptional inhibition of the CCNE1 expression.
Reciprocal results were observed in experiments carried out
in HeLa cells using 2'-O-Methylated anti-miR-16-1 (Fig. 3c),
which binds to endogenous miR-16-1 and thereby antagonizes
its activity. Taken together, these data suggest that miR-16-1
directly binds to the 3’ UTR of CCNE1 and downreguates its
expression.
miR-16-1 induces G0/G1 cell cycle arrest partially through
down-regulation of CCNE1
Since CCNE1 has a prominent role in cell cycle, particularly
drives cells progressing from G1 to S phase, we then examined
if miR-16-1 regulation of CCNE1 expression correlates with
cell cycle regulation. A549 human lung carcinoma cells were
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transfected with synthetic miR-16-1 duplex or siRNAs against
CCNE1, then the cell cycle distribution of the transfected cells
treated with microtubule depolymerizing drug nocodazole 24
hours post-transfection were analyzed by flow cytometry.
Compared with cells transfected with negative control (NC)
duplex, cells transfected with siRNA against CCNE1 triggered
an accumulation of cells at the G0/G1 stage, whereas the
numbers of cells in S-phase and G2/M-phase accordingly
decreased (Fig. 4a), which consistent with the role of CCNE1
in cell cycle progression from G1 to S phase. As predicted for
downregulation of CCNE1 expression by miR-16-1, transfection with miR-16-1 yielded a phenocopy of the phenotype
generated by siRNA against CCNE1, and the effects were more
profound, featured by a greater G0/G1-cell accumulation and
a greater decrease in cell population in S-phase and G2/Mphase (Fig. 4a). Comparable results were also observed in
human breast cancer MCF-7 cells (Supplementary Fig. 1).
One explanation for the more profound cell cycle arrest at
G0/G1 phase elicited by miR-16-1 than siRNA against CCNE1
is that multiple cell cycle genes coordinating cell cycle
progression are targeted by miR-16-1. CCNE1 is only one of
such targets regulated by miR-16-1 in modulating cell cycle
progression.
To further establish a functional connection between miR16-1 and CCNE1, we addressed whether high level of CCNE1
is account for the cell cycle phenotype caused by the reduced
level of miR-16-1. Anti-miR-16-1 was used to reduce the miR16-1 activity in A549 human lung carcinoma cells. As shown
in Fig. 4b, compared to control-treated cells, transfection with
anti-miR-16-1 resulted in a reduced level of G0/G1-cells and a
correspondingly accumulation of cells in S phase. If the antimiR-16-1-induced phenotype depends on increased levels of
CCNE1, then reducing the level of CCNE1 should abrogate the
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microRNA-16-1 downregulates cyclin E1 expression
Fu Wang, et al.
Fig. 4. Ectopic miR-16-1 triggers G0/G1 arrest
in A549 cells, and si-CCNE1 rescues the antimiR-16-1-induced phenotype. (a) A549 cells
were transfected with synthetic miR-16-1 duplex, siRNA against CCNE1 (si-CCNE1) or negative control (NC) duplex, 24 hr post-transfection, cells were then treated with nocodazole for 16∼20 hr. Cell cycle distribution were
analyzed by flow cytometry. (b) A549 cells
were transfected anti-miR-16-1 and negative
control (NC), anti-miR-16-1 and si-CCNE1, or
NC and scramble anti-miR control (SC). The
cells were collected 48 hr after transfection
and subjected to flow cytometry analysis. In
each instance, flow cytometry was performed
three times, the shown data represent three
independent experiments.
effect. To test this hypothesis, anti-miR-16-1 was cotransfected
with siRNA against CCNE1. The result showed that si-CCNE1
led to substantial, although not complete, rescue of anti-miR16-1-induced cellular phenotype (Fig. 4b). These data suggeste
that CCNE1 is required for the anti-miR-16-1 phenotype and
indicate for further that CCNE1 is only one of the targets
modulated by miR-16-1 in cell cycle regulation. Overall, these
results show that miR-16-1 contributes to induction of G0/G1
arrest in A549 cells and MCF-7 cells, which is partially
through down-regulation of CCNE1.
DISCUSSION
MicroRNAs are of ever increasing importance as post-transcriptional regulators of gene expression following transcription.
They have been demonstrated to play a significant role in
carcinogenesis by altering expression of oncogenes and tumor
suppressor genes (15). In this study, we have confirmed by
multiple methods that miR-16-1 controls the expression of a
positive cell cycle regulator CCNE1 oncogene by directly
targeting the 3’ UTR of its mRNA, which reveals a mechanism
of post-transcriptional regulation of CCNE1 expression and
connects the function of miR-16-1 with a cell cycle gene.
Our bioinformatic analysis has suggested that in addition to
miR-16-1, miR-195 in miR-16 family and some other miRNAs
may also regulate the expression of CCNE1 at post-transcriptional level. Meanwhile, it is likely that CCNE1 is only one of
the multiple targets coordinately regulated by miR-16-1 in
controlling cell cycle progression. This can be reflected by the
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findings that siRNA against CCNE1 only partially phenocopied
the cellular phenotype of miR-16-1 and incompletely rescued
the anti-miR-16-1-induced phenotype (Fig. 4). An emerging
common theme is that microRNAs modulate cell cycle progression by a coordinated repression of multiple genes. In
other words, multiple targets regulated by a single miRNA can
act in concert, rather than individually, to regulate the same
biological process. Coordinated regulation of many targets by
a single miRNA may allow for a prompt cellular response to
the progression of the cell cycle and also for rapid reversal of
the microRNA-induced cell cycle regulation upon changes in
miRNA synthesis, stability or localization (27).
We found that miR-16-1 also triggered G0/G1 arrest in
MCF-7 breast cancer cells, but led to a less G0/G1 accumulation phenotype as compared to that in A549 lung carcinoma
cells (supplementary Fig. 1). One possible explanation for this
finding is that the genetic context in individual cell line is
distinct from the other, for example, the expression profile of
the targeted genes of miR-16-1 differs in these two cell lines.
The present study helps to further our understanding of the
molecular pathways involved in the development and progression of lung carcinoma and breast cancer, which may
implicate new therapeutic strategies in the treatment of these
diseases. MiR-16-1 could negatively regulate CCNE1 oncogene
overexpressed in the development of a subset of human
cancers, and thus has a strong rationale for cancer therapy in
the future.
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microRNA-16-1 downregulates cyclin E1 expression
Fu Wang, et al.
MATERIALS AND METHODS
Cell lines and transfection
HEK293 and HeLa cell lines were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine
serum (FBS) and 100 U/ml penicillin/streptomycin (Gibco) at
o
37 C in a humified atmosphere of 5% CO2. MCF-7 and A549
cells were grown at the same condition except that the fetal
bovine serum (FBS) was substituted for the 10% new-born calf
serum (Gibco). Transfections were performed by Lipofectamine 2000 reagent (Invitrogen) for plasmids or Oligofectamine
reagent (Invitrogen) for RNA oligos according to the manufacturer’s protocol.
Plasmids and RNA oligos
A miRNA construct expressing miR-16-1 was designed by our
laboratory. In brief, a genomic fragment spanning the miR16-1 locus from human chromosome 13 was cloned into the
Xho I/Sac II restriction site of a mammalian expression vector
pZW8. The PCR primers were used as follws: miR-16-1 FW
5'-CCG CTC GAG TGC AGG CCA TAT TGT GCT GCC-3' and
miR-16-1 RV 5'-TCC CCG CGG ATT GTC TTC TAA GCT CTG
TTC-3'. The full length 3’ UTR of CCNE1 was amplified from
HeLa cell genomic DNA by PCR using the following primers:
CCNE1-UTR FW 5'-CTA GTC TAG ACC ACC CCA TCC TTC
TCC A-3' and CCNE1-UTR RV 5'-CTG GGG GCC CTG TCT
CAA AAA CAG TAT TAT CTT-3' and inserted at the XbaI and
Apa I site, immediately downstream of the luciferase gene in
the modified pRL vector (Promega). Mutant 3’ UTRs were
generated by overlap-extension PCR method by the following
primers: CCNE-MUT1 FW 5’-CGA TAC CAT GGA AGG TGC
TAC TTG ACC T-3’, CCNE-MUT1 RV 5’-TCG GGA GCA CGC
ACT GGT-3’; CCNE-MUT2 FW 5’-CGA TAC CAT ATC TAT
CCA TTT TTT AAT AAA GAT A-3’, CCNE-MUT2 RV 5’-CTT
ACA AAA CAG TTC ATC AAA GG-3’. Wild type and mutant
inserts were confirmed by sequencing. Synthetic RNA oligos
were synthesized and purified by GenePharma Co. (Shanghai,
China), the sequences were：miR-16-1 mimics (sense: 5'-UAG
CAG CAC GUA AAU AUU GGC G-3', anti-sense: 5’-CGC
CAA UAU UUA CGU GCU GCU A-3’), negative control
(sense: 5’-UUG UAC UAC ACA AAA GUA CUG-3’, antisense: 5’-CAG UAC UUU UGU GUA GUA CAA-3’), 2'-OMethylated anti-miR-16-1 inhibitor （5'-CGC CAA UAU UUA
CGU GCU GCU A-3'), scramble anti-miR control (5’-UUG
UAC UAC ACA AAA GUA CUG-3’)， siRNA against CCNE1
(sense: 5’-CAC CCU CUU CUG CAG CCA A dTdT-3’,
anti-sense: 5’-UUG GCU GCA GAA GAG GGU G dTdT-3’)
RNA extraction and qRT-PCR
Total RNA was extracted from the cultured cells using Trizol
Reagent (Invitrogen) according to the manufacturer’s protocol.
qRT-PCR was used to confirm the expression level of mRNAs.
cDNA was produced with random primers and reverse
transcription was performed according to the protocol of MMLV Reverse Transcriptase (Promega), and qPCR was performed as described in the method of SYBR Green Realtime
PCR Master Mix (ToYoBo, Japan) with Rotor-Gene 3000 Mutifilter Real-time Cycler detection system (Corbett Research, Sydhttp://bmbreports.org
ney, Australia) supplied with analytical software. The PCR
o
reaction was conducted at 95 C for 5 min followed by 40
o
cycles of 95 C for 30 s and 60oC for 30 s. GAPDH mRNA
levels were used for normalization. The oligonucleotides used
as PCR primers were: GAPDH FW, 5’-ACC ACA GTC CAT
GCC ATC AC-3’, RV, 5’-TCC ACC ACC CTG TTG CTG TA-3’.
CCNE1 FW, 5’- CCA CAC CTG ACA AAG AAG ATG ATG
AC-3’, RV, 5’-GAG CCT CTG GAT GGT GCA ATA AT-3’
Western blot
Cells were lysed then the lysates were boild for 5 min and
centrifuged at 12,000 r/min at 4oC for 10 min. The whole cell
lysate of 40 μg was loaded per lane and separated using 12%
SDS-acrylamide gels, and transferred to nitrocellulose membranes (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk in TBS, the membrane was probed with primary
monoclonal antibody specific to CCNE1 (1 : 1,000, Eptomics,
CA) or β-actin (1 : 10,000, Sigma), which was used as an
internal control for protein loading. The membrane was further
probed with horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG (1 : 10,000, Sigma) and the protein bands were
visualized using enhanced chemiluminescence detection
reagents (Pierce).
Luciferase activity assay
For luciferase analysis, HEK293 or HeLa cells were transiently
transfected with 200 ng of each renilla luciferase reporter
plasmid plus 40 ng pGL3-promoter (Promega), in combination
with 50 nM of synthetic miR-16-1 duplex or 2’-O-Methylated
anti-miR-16-1 using Lipofectamine 2000 (Invitrogen) according
to the manufacturer’s protocol. Luciferase activity was measured 24 hr after transfection using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Three independent experiments were performed in triplicate.
Flow cytometry
Flow cytometry analysis was done as described previously (28,
29) but with some modifications. Briefly, one day before transfection, equal numbers of A549 cells or MCF-7 cells (2.0 ×
4
10 ) were seeded into 12-well tissue culture plates without
antibiotics so they will be about 30% confluent at the time of
transfection. Next day cells were transiently transfected with
negative control siRNA or miR-16-1 duplex or siRNA against
CCNE1 at a final concentration of 50 nM using Oligofectamine (Invitrogen). Twenty-four hours after transfection, nocodazole (100 ng/ml; Sigma-Aldrich) was added and cells were
further incubated for 16 to 20 h before harvesting. The cells
were collected by centrifugation, fixed with ice-cold 70%
o
ethanol at -20 C, washed with phosphate-buffered saline (PBS),
and resuspended in 0.5 ml of PBS containing propidium
iodide (50 μg/ml) and RNase A (1 mg/ml). After a final incubation at 37°C for 30 min, cells were analyzed by the EPICS
ALTRA II flow cytometer (Beckman Coulter). A total of 10,000
events were counted for each sample. Data were analyzed
using Muticycle AV software (Beckman Coulter).
Statistical analysis
Results were expressed as means ± SD unless indicated otherBMB reports
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microRNA-16-1 downregulates cyclin E1 expression
Fu Wang, et al.
wise. Differences between groups were assessed by unpaired,
two-tailed Student’s t-test, p ＜ 0.05 was considered significant.
All data were plotted using the GraphPad Prism 4.0 program
(www.graphpad.com).
Acknowledgements
We would like to gratefully thank Yan Wang and Weihuang Liu
(Medical college, Wuhan University, China) for the flow cytometry technical assistance and data analysis. This work is supported
by National High-tech R&D Program (863 Program) of China
through grant 2007AA02Z100 awarded to Y. Zhang and by the
National Basic Research Program (973) of China through grant
2005CB724604 awarded to Y. Zhang and X.-D. Fu.
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