An estrogen receptor a suppressor, microRNA-22, is
downregulated in estrogen receptor a-positive human
breast cancer cell lines and clinical samples
Jianhua Xiong
1,
*, Dianke Yu
2,
*, Na Wei
1
, Hanjiang Fu
3
, Tianjing Cai
1
, Yuanyu Huang
1
, Chen Wu
2
,
Xiaofei Zheng
3
, Quan Du
1
, Dongxin Lin
2
and Zicai Liang
1
1 Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, China
2 Department of Etiology and Carcinogenesis, State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing, China
3 Beijing Institute of Radiation Medicine, China
Introduction

January 2010, accepted 25 January 2010)
doi:10.1111/j.1742-4658.2010.07594.x
Previous studies have suggested that microRNAs (miRNAs) may play
important roles in tumorigenesis, but little is known about the functions of
most miRNAs in cancer development. In the present study, we set up a
cell-based screen using a luciferase reporter plasmid carrying the whole
 4.7 kb 3¢-UTR of estrogen receptor a (ERa) mRNA cotransfected with
a synthetic miRNA expression library to identify potential ERa-targeting
miRNAs. Among all the miRNAs, miR-22 was found to repress robustly
the luciferase signal in both HEK-293T and ERa-positive MCF-7 cells.
Mutation of the target site was found to abrogate this repression effect of
miR-22, whereas antagonism of endogenous miR-22 in MDA-MB-231 cells
resulted in elevated reporter signals. We assessed the miR-22 expression
patterns in five breast cancer cell lines and 23 clinical biopsies and revealed
that there is a significant inverse association between the miR-22 levels and
ERa protein expression. To evaluate the potential of miR-22 as a potential
therapeutic intervention, we found that reduction of endogenous ERa pro-
tein levels and suppression of cancer cell growth could be achieved in
MCF-7 cells by miR-22 overexpression in a way that can be recapitulated
by the introduction of specific small interfering RNA against ERa. The
phenomena can be rescued by the reintroduction of ERa. Taken together,
our data indicate that miR-22 was frequently downregulated in ERa-posi-
tive human breast cancer cell lines and clinical samples. Direct involvement
in the regulation of ERa may be one of the mechanisms through which
miR-22 could play a pivotal role in the pathogenesis of breast cancer.
Abbreviations
DMEM, Dulbecco’s modified Eagle’s medium; ERa, estrogen receptor a; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
miRNA, microRNA; siRNA, small interfering RNA.
1684 FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS
frequencies of amplification or deletion, and frequent

16]. However, the functional roles of most miRNAs in
the development of breast cancer remain unknown.
In this study, we identified miR-22 as a potent regu-
lator of ESR1 encoding estrogen receptor a (ERa) and
demonstrated that miR-22 is frequently downregulated
in ERa-positive human breast cancer cell lines and
clinical samples. In addition, further functional
studies showed that ERa plays an important role in
miR-22-mediated growth retardation of tumor cells.
Results
Identification of miRNAs that might target ERa
3¢-UTR
To identify human miRNAs that might target ERa
3¢-UTR, we used the targetscan program (http://
www.targetscan.org/) to predict miRNAs that have the
interaction with 3¢-UTR of ESR1 mRNA. Along the
 4.3 kb full length of 3¢-UTR of ESR1 mRNA, 59
miRNAs had conserved target sites and partial
miRNA families broadly conserved among vertebrates
were enumerated according to their conserved target
positions (Fig. 1A). In addition, miR-206, which has
two target sites on 3¢-UTR of ESR1 mRNA, was pre-
viously reported as a negative regulator of ERa [17].
miR-9 and miR-1 were implicated in crucial cancer-
related cell signaling regulation [18,19]. The 62
miRNAs were chosen as our preferred candidates
for ERa regulators. To evaluate comprehensively
miRNAs–ESR1 mRNA interactions, we used the
screening system based on a luciferase reporter plasmid
carrying the full-length 3¢-UTR of ESR1 mRNA. As a

We therefore constructed a reporter plasmid
(pGL3m–ESR1–3¢-UTR–WT) with the 4.3 kb ESR1
3¢-UTR cloned downstream to a firefly luciferase
reporter gene and used both vector-expressed miR-22
and synthetic miR-22 to evaluate the suppression
effects of the miRNAs on the reporter gene expression.
It was found that in HEK293T and ERa-positive
MCF-7 cells, miR-22 had a potent inhibitory effect on
the expression of the reporter gene with the ESR1
3¢-UTR tag (Fig. 2B, C). To examine whether the ERa
silencing is mediated by specific and direct interaction
of miR-22 with the ESR1 target site, the complemen-
tary site for the miR-22 seed region was mutated to
J. Xiong et al. MicroRNA-22 and estrogen receptor a
FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1685
form pGL3m–ESR1–3¢-UTR–MUT (Fig. 2A). Both
pcDNA3.0–miR-22 and miR-22 duplex reduced lucif-
erase activities expressed in pGL3m–ESR1–3¢-UTR–
WT by  50%, but such a reduction was completely
abolished in pGL3m–ESR1-3¢–UTR–MUT (Fig. 2B,
C). Moreover, knockdown of endogenous miR-22 in
MDA-MB-231 cells that express a relatively high level
miR-22 could elevate the luciferase signal of pGL3m–
ESR1–3¢-UTR–WT (Fig. 2D), further suggesting that
silencing of ER a was indeed by the interaction of
miR-22 with the 3¢-UTR of ESR1.
The effect of miR-22 on endogenous ERa protein
levels was also examined in MCF-7 and MDA-MB-
231 cells. The results showed that an ectopic increase
in either synthesized or vector-expressed miR-22 in

was set as 100%. Data are presented as
mean ± standard deviation from at least
three independent experiments.
MicroRNA-22 and estrogen receptor a J. Xiong et al.
1686 FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS
AB
CD
GFE
Fig. 2. Direct regulation of ERa expression by miR-22. (A) A putative miR-22-binding target region in the 3¢-UTR of ESR1 mRNA among
mammalian species (upper panel, shown in red); site-direct mutations in the sequence complimentary to the seed region for miR-22 (lower
panel, shown in red). (B–D) Relative luciferase activity of pGL3m–ESR1–3¢-UTR–WT (ESR1–3¢-UTR–WT) and pGL3m–ESR1–3¢-UTR–MUT
(ESR1–3¢-UTR–MUT) in HEK293T and MCF-7 cells cotransfected with pcDNA-3.0–miR-22 or pcDNA-3.0 and synthetic miR-22 duplex or con-
trol RNA duplex,and in MDA-MB-231 cells with anti-miR-22 or control anti-miR. Relative luciferase activity was measured 48 h after transfec-
tion and normalized by Renilla luciferase activity generated by cotransfected pRL-TK vector. The normalized luciferase activity for the
controls was set as 1. Data are presented as mean ± standard deviation from at least three independent experiments (**P < 0.01). (E) Sup-
pression of ERa expression in MCF-7 cells by pcDNA-3.0–miR-22 or synthetic miR-22 duplex. MCF-7 cells were harvested 48 h after trans-
fection and cell lysate was applied to a western blot. b-actin was used as a loading control and the relative density of bands was
densitometrically quantified. (F) Upregulation of ERa expression in MDA-MB-231 cells by anti-miR-22. MDA-MB-231 cells were harvested
48 h after transfection and cell lysate was applied to a western blot. b-actin was used as a loading control. (G) Relative level of ERa mRNA
was detected using quantitative RT-PCR with GAPDH as an internal control.
J. Xiong et al. MicroRNA-22 and estrogen receptor a
FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1687
Frequent downregulation of miR-22 expression in
ERa-positive breast cancer cell lines and clinical
samples
To evaluate the therapeutic potential and to extend the
mechanistic insight of miR-22 as an ERa suppressor,
we measured its expression levels using quantitative
RT-PCR in five breast cancer cell lines that are either
ERa positive (MCF-7, T-47D and BT-474) or ERa

breast cancer cell lines.
ERa is potentially involved in miR-22-mediated
repression of ERa-positive breast cancer cell
growth
To investigate the role of ERa in miR-22-mediated
repression of human cancer cell growth of ERa-posi-
tive breast cancer cells we used two specific small inter-
fering RNAs (siRNA) against ERa. MCF-7 cells were
transfected with ERa siRNAs or control RNA duplex.
After incubation for 48 h, the expression of ERa
was subjected to quantitative RT-PCR detection or
A
B
C
Fig. 3. Frequent downregulation of miR-22 expression in ERa-posi-
tive breast cancer cell lines and tumor specimens. (A) For breast
cancer cell lines, the expression levels of mature miR-22 were
determined by quantitative RT-PCR with U6 as an internal standard.
miR-22 expression levels are presented as mean ± standard devia-
tion from at least three independent experiments. The P values of
comparisons between two groups are as follows: P = 0.0018, com-
parison between MCF-7 and MDA-MB-231; P = 0.0012, compari-
son between T-47D and MDA-MB-231; P = 0.0010, comparison
between BT-474 and MDA-MB-231; P = 0.0399, comparison
between MCF-7 and SK-BR-3; P = 0.0123, comparison between
T-47D and SK-BR-3; P = 0.0074, comparison between BT-474 and
SK-BR-3. (B) The ERa expression status of breast cancer cell lines
was examined by western blotting. (C) For breast cancer speci-
mens, miR-22 expression data are illustrated using a box plot. The
line inside each box is the median; the upper and lower limits of

identified using experiment-driven methods and compu-
tation-driven approaches [24,25]. miRNAs have diverse
expression patterns in different cell types and it is well
accepted that miRNAs regulate numerous physiological
and pathological processes [1,26]. The biological func-
tion of most miRNAs is, however, largely unknown.
miRNAs have been relatively better investigated in
tumor cells and it has already been shown that
miRNAs can function as both tumor suppressors and
oncogenes by directly regulating genes involved in
related pathways. Unrestrained cell proliferation and
deregulated cell death underlie neoplastic progression in
almost all cancer types [13,27]. An increasing number of
miRNAs have been implicated in tumorigenesis via the
regulation of cancer cell proliferation and growth. For
instance, let-7 can inhibit proliferation of lung and liver
cancer cells by targeting multiple cell cycle oncogenes
[28] and miR-34b and miR-34c have a cooperative
negative effect on proliferation and colony formation
of ovarian cancer cells [6], whereas overexpression of
the miR-17-92 cluster miRNAs enhance lung cancer
cell proliferation and growth as oncogenes [29].
The highly conserved human miR-22 gene is located
at a fragile cancer-relevant genomic region in chromo-
some 17 (17p13.3), and mapped to an exon of the
C17orf91 gene [4,30]. To date, several genes, including
HOXA6, HOXA4, HSPG2, GPNMB, CLIC4 and SP1,
have been predicted as targets of miR-22 [31–33],
whereas ERa has been suggested as a direct target of
this miRNA in a recent work [34]. miRNA expression

significantly elevated by knockdown of endogenous
miR-22 in MDA-MB-231 cells that expressed a rela-
tively high level of miR-22. These results demonstrate
that ERa is a direct target of miR-22. miR-22 treatment
was found to dramatically reduce the endogenous trans-
lational yield of ERa, and knockdown of endogenous
miR-22 could elevate ERa protein expression.
Because ERa expression is routinely monitored in
breast cancer samples as a prognostic marker, we went
further to assess the correlation between miR-22
expression and ERa protein levels in breast cancer cell
lines and surgical specimens. It was interesting to find
that downregulation of miR-22 expression occurs fre-
quently, not only in ERa-positive human breast cancer
cell lines, but also in surgical specimens compared with
ERa-negative counterparts. This result made it appeal-
ing to examine whether miR-22 could also be used as
a marker for the identification of breast cancer sub-
types in addition to ERa itself, as miR-22 probably
regulates a different set of genes in comparison with
the regulatory profile of ERa.
J. Xiong et al. MicroRNA-22 and estrogen receptor a
FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1689
A
C
D
B
Fig. 4. ERa is potentially involved in miR-22-mediated repression of human ERa-positive breast cancer cell growth. (A) Regulation of ERa
expression by two ERa siRNAs. MCF-7 cells were transfected with control RNA duplex or ERa siRNAs, total RNAs were prepared and ana-
lyzed for ERa mRNA expression by quantitative RT-PCR at 48 h after transcription. The data were normalized against the expression of

(American Type Culture Collection, Manassas, VA, USA)
and MCF-7 cells were maintained in 10% fetal bovine
serum-supplemented Dulbecco’s modified Eagle’s medium
(DMEM) (Hyclone, Logan, UT, USA); MDA-MB-231 cells
were maintained in 10% fetal bovine serum-supplemented
L-15 (Gibco, Grand Island, NY, USA); SK-BR-3 cells were
maintained in 10% fetal bovine serum-supplemented
RPMI-1640 (Hyclone); T-47D and BT-474 cells were
maintained in RPMI-1640 (Hyclone) plus 10% fetal bovine
serum and 0.2 UÆmL
)1
insulin.
Vector construction
A DNA segment encompassing the mature miR-22
sequence and its 5¢- and 3¢-flanking regions (130 and
80 bp, respectively) was cloned into the BamHI and XhoI
sites in pcDNA3.0 (Invitrogen, Carlsbad, CA, USA) to
create the miR-22 expression vector pcDNA3.0–miR-22.
pGL3m was modified from a firefly luciferase-expressing
vector pGL3-control (Promega, Madison, WI, USA) by
inserting a multiple cloning sequence downstream of the
XbaI site, including EcoRV, ApaI, SacII, NdeI, PstI,
EcoRI and NruI sites. The insertion site is immediately
downstream of the stop codon of the firefly luciferase
reporter gene. A 4.3 kb fragment encoding the full-length
3¢-UTR of human ESR1 mRNA (Genbank accession no.
NM_000125) was cloned between the SacII and EcoRI
sites in pGL3m, forming pGL3m–ESR1–3¢-UTR–WT, in
which, site-specific mutations were performed to disrupt
the binding site of miR-22, forming pGL3m–ESR1–3¢-

tion. The anti-miR-22 was a 2¢-O-methyl-modified oligori-
bonucleotide designed as an inhibitor of miR-22, and its
sequence is 5¢-ACAGUCUUCAACUGGCAGCUU-3¢. The
negative control for anti-miR-22 in the antagonism experi-
ments was control anti-miR, with a sequence of 5¢-GUG
GAUAUUGUUGCCAUCA-3¢. The sequences of two siR-
NAs for ESR1 are as follows: ERa siRNA #2 sense strand
5¢-UCAUCGCAUUCC UUGCAAAdTdT-3 ¢, antisense
strand 5¢- UUUGCAAGGAAUGCGAUGAdTdT-3¢;ERa
siRNA #3 sense strand 5¢- GGAGAAUGUUGAAACA
CAAdTdT-3¢, antisense strand 5¢- UUGUGUUUCAA
CAUUCUCCdTdT-3¢. The transfection efficiency was
monitored by fluorescence-activated cell sorting, using a
carboxyfluorescein (FAM)-labeled siRNA.
Western blot
Forty-eight hours after transfection, the cells were lysed
using cell lysis buffer (Cell Signaling Technology, Beverly,
MA, USA). Isolated proteins were separated in 10% SDS
polyacrylamide gels, transferred to polyvinylidene difluoride
membranes (Bio-Rad Laboratories, Hercules, CA, USA),
and detected with antibodies for human ERa (Cell Signal-
ing Technology), b-actin (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) and ECL kit (Santa Cruz Biotechnology).
The intensity of protein bands was quantified using image j
software (National Institutes of Health, Bethesda, MD,
USA).
J. Xiong et al. MicroRNA-22 and estrogen receptor a
FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1691
Luciferase reporter assay
For miRNA duplex library analysis, HEK293T and MCF-7

and then converted to cDNA using miR-22 RT primer
(Ribobio) and ImPro-II reverse transcriptase (Promega).
Soft-agar colony assay
Anchorage-independent growth was carried out in six-well
plates coated with 0.6% soft agar in DMEM plus 10% fetal
bovine serum. Twenty-four hours after transfection, 1 · 10
4
transfected cells were plated into each well of six-well plates
and maintained in DMEM plus 10% fetal bovine serum for
2 weeks. Colonies were stained with 1.25 mgÆmL
)1
nitroblue
tetrazolium for 16 h before imaging.
Statistical analysis
Data are presented as mean ± standard deviation from at
least three independent experiments and differences were
assessed using Student’s t test. The Kruskal–Wallis one-way
analysis of variance test was used to test the significance of
association between ERa status and the levels of miR-22 in
tumor specimens. These statistical analyses were imple-
mented in statistic analysis system software (version 8.0,
SAS Institute). P < 0.05 was used as the criterion for
statistical significance; all statistical tests were two-sided.
Acknowledgements
We thank Dr Yangming Wang for critical reading of
the manuscript. This work was supported by
the National High-tech R&D Program of China
(2007AA02Z165, 2008DFA30770), the National Basic
Research Program of China (2007CB512100), and the
National Foundation of Natural Science (grant

‡ 50 years 7 (70.0) 7 (53.8)
Tumor size
£ 2 cm 5 (50.0) 7 (53.8)
> 2 cm 5 (50.0) 6 (46.2)
Lymph node metatasis
No 4 (40.0) 8 (61.5)
Yes 6 (60.0) 5 (38.5)
Histology
Invasive ductal carcinoma 10 (100) 12 (92.3)
Medullary carcinoma 0 (0) 1 (7.7)
MicroRNA-22 and estrogen receptor a J. Xiong et al.
1692 FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS
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Supporting information