An estrogen receptor α suppressor, microRNA22, is downregulated in estrogen receptor α-positive human breast cancer cell lines and clinical samples

An estrogen receptor a suppressor, microRNA-22, isdownregulated in estrogen receptor a-positive humanbreast cancer cell lines and clinical samplesJianhua Xiong1,*, Dianke Yu2,*, Na Wei1, Hanjiang Fu3, Tianjing Cai1, Yuanyu Huang1, Chen Wu2,Xiaofei Zheng3, Quan Du1, Dongxin Lin2 and Zicai Liang1

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

MicroRNAs (miRNAs), a class of endogenous short

(� 22 nucleotides) noncoding RNAs, have been

reported to be capable of suppressing the expression of

protein-coding genes at the post-transcriptional level

by cleaving target mRNAs and ⁄or repressing their

translation [1]. Aberrant expression of miRNAs is

known to be involved in various human diseases,

including cancer [2,3]. In two recent studies, more than

50% of human miRNA genes have been mapped to

the cancer-related chromosomal regions with high

Keywords

breast carcinoma; estrogen receptor a;

microRNA-22; proliferation

Correspondence

Z. Liang, Laboratory of Nucleic Acid

Technology, Institute of Molecular Medicine,

Peking University, Beijing 100871, China

Fax: +86 10 62769862

Tel: +86 10 62769862

E-mail: [email protected]

Dongxin Lin, Department of Etiology and

Carcinogenesis, Cancer Institute, Chinese

Academy of Medical Sciences, Beijing

100021, China

Fax: +86 10 67722460

Tel: +86 10 87788491

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 30 September 2009, revised 5

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-targetingmiRNAs. 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

genomic alterations of miRNAs were observed in can-

cers [4,5]. Emerging evidence shows that miRNAs

function as oncogenes or tumor suppressors to modu-

late multiple oncogenic cellular processes, including

cell proliferation, apoptosis, invasion and migration

[6–8]. For example, it has been shown that p53-depen-

dent miR-34b and miR-34c cooperate to inhibit the

proliferation of neoplastic epithelial ovarian cells [6],

and miR-15 and miR-16 simulate apoptosis in chronic

lymphocytic leukemia by targeting BCL2 [7]. On the

other hand, miR-10b initiates breast cancer invasion

and metastasis by repressing homeobox D10 [8]. More-

over, miRNA expression profiling has been used as a

signature to distinguish different cancer types and to

provide an accurate classification of poorly differenti-

ated tumors [9]. In view of the roles that miRNAs play

in human diseases, including cancer, miRNAs have

been considered to be potential drug candidates or

therapeutic targets [10].

Breast cancer is one of the most common and preva-

lent cancers in women and a leading cause of cancer-

related death [11]. As in other common cancers, the

formation and progression of breast cancer is a multi-

step process involving genetic and epigenetic altera-

tions that drive unrestrained cell proliferation and

growth [12,13]. Several aberrantly expressed miRNAs

have been identified in breast cancer, such as miR-21,

miR-205 and miR-17-5p, which were shown to be

involved in the formation of breast cancer by targeting

the TPM1, HER3 and AIB1 genes, respectively [14–

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) anddemonstrated 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 ERa3¢-UTR

To identify human miRNAs that might target ERa3¢-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

result, nine miRNAs were found to suppress the

expression of the reporter by more than 40%, and 25

miRNAs were found to suppress the expression of the

reporter by more than 20% (Fig. 1B). This might

constitute the major category of miRNAs that play

regulatory roles on ERa through interactions with

3¢-UTR of ESR1 mRNA. As indicated in Fig. 1B,

miR-22 could induce an �40% reduction in the

luciferase signal.

Direct regulation of ERa expression by miR-22

We compared three popular miRNA target prediction

programs and found that miR-22 was highly scored in

all three algorithms [targetscan, miranda (http://

www.microrna.org/microrna/home.do) and pictar

(http://pictar.mdc-berlin.de/)] for targeting ESR1

(Table S1) [20,21]. The targetscan prediction sug-

gested that ESR1 has an extremely conserved miR-22

target site (position 2292–2298 of human ESR1

3¢-UTR) in human and other mammalian species,

including the chimpanzee, rhesus monkey, mouse, rat,

dog and rabbit (Fig. 2A). The predicted DG of 70 bp

5¢- and 3¢-flanking regions of neighboring potential

conserved miR-22 target site was determined by mfold

and the resulting DG values ()10.70 and )8.40kcalÆmol)1, respectively) suggested that miR-22 may

have access to its conserved target ESR1 mRNA site

[22,23].

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-positiveMCF-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 ERasilencing 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 ERa 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

MCF-7 led to an �50% reduction in ERa protein

levels (Fig. 2E). Conversely, ERa expression was sig-

nificantly elevated by inhibiting endogenous miR-22 in

MDA-MB-231 cells (Fig. 2F). Interestingly, the reduc-

tion in ERa protein levels was markedly greater than

the reduction in ESR1 mRNA levels determined by

quantitative RT-PCR (Fig. 2G). These results demon-

strated that miR-22 could regulate ERa expression by

directly binding to ERa 3¢-UTR, and inhibited ERaexpression through both destabilizing mRNA and

inhibiting translation.

A

B

Fig. 1. Identification of miRNAs that might

target ERa 3¢-UTR. (A) The 59 miRNAs

predicted as having broadly conserved sites

by the TARGETSCAN program. Partial miRNA

families broadly conserved among verte-

brates were enumerated according to their

conserved target positions. (B) The effects

of the predicted 59 miRNAs as well as three

interested miRNAs (miR-206, miR-9 and

miR-1) on reporter gene expression of

pGL3m–ESR1–3¢-UTR–WT. Relative lucifer-

ase activity was measured 48 h after

transfection and normalized by Renilla

luciferase activity generated by

cotransfected pRL-TK vector. The

normalized luciferase activity for the controls

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.


A B

C D

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.



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 ERanegative (MDA-MB-231 and SK-BR-3), and 23 breast

tumor specimens, of which 10 are ERa positive and 13

are ERa negative.

Among all breast cancer cell lines examined, miR-22

expression was found to be significantly lower in ERa-positive lines, such as MCF-7 (2.290 ± 0.499), T-47D

(1.573 ± 0.325) and BT474 (1.152 ± 0.318), than

in ERa-negative lines, such as MDA-MB-231

(10.732 ± 1.923) and SK-BR-3 (4.269 ± 1.027). The

differences were determined by Student’s t-test as

P = 0.0018 for the comparison between MCF-7

and MDA-MB-231, P = 0.0012 for the comparison

between T-47D and MDA-MB-231 and P = 0.0010

for the comparison between BT-474 and MDA-MB-

231, but P = 0.0399 for the comparison between

MCF-7 and SK-BR-3, P = 0.0123 for the comparison

between T-47D and SK-BR-3 and P = 0.0074 for the

comparison between BT-474 and SK-BR-3 (Fig. 3A).

The ERa expression status of breast cancer cell lines

was confirmed using immunoblotting (Fig. 3B).

For breast cancer clinical samples, ERa-positivebreast tumor specimens had significantly lower miR-22

levels (0.913 ± 0.807, range 0.112–2.10) than ERa-negative specimens (2.410 ± 2.550, range 0.615–9.64;

P = 0.044; Fig. 3C). As indicated, the P value was

0.044; the Kruskal–Wallis one-way analysis of variance

test indicated that the levels of miR-22 were inversely

associated with ERa expression status in tumor speci-

mens, which is in good agreement with the inverse cor-

relation between the expression of miR-22 and ERa in

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 ERawas 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

the box are the 75th and 25th percentiles, respectively, and the

vertical bars above and below the box indicate the maximum and

minimum values. The solid circles are outlier values. The expres-

sion levels of mature miR-22 were determined by quantitative

RT-PCR with U6 as an internal standard (*P = 0.044).



immunoblot analysis. The result showed that the two

siRNAs could reduce ERa mRNA and protein level

significantly (Fig. 4A,B). Further functional studies

showed that knockdown of ERa by the two siRNAs

could mimic the inhibitory effect of miR-22 on the

proliferation and colony formation of breast cancer

cells, whereas a control siRNA duplex did not show

an effect (Fig. 4C,D).

We then went further to determine whether overex-

pression of ERa could counterbalance the antigrowth

effect of miR-22 on MCF-7 cells. We forced MCF-7

cells to express ERa constitutively using a construct

encoding the entire encoding region of ERa mRNA, but

lacking the ERa 3¢-UTR, thus yielding an mRNA that

is resistant to miR-22-mediated inhibition of translation.

Indeed, we found that the miR-22-induced cell growth

repression phenotype was partially rescued by the intro-

duction of this vector expressing an miRNA-resistant

ERa transcript (Fig. 4C, D). These findings suggest that

ERa plays an important role in miR-22-retarded growth

of ERa-positive breast cancer cells.

Discussion

To date, more than 700 human miRNAs have been

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

profiling data revealed that miR-22 had a great reduc-

tion in acute myeloid leukemia with mutations in

NPM1 compared with acute myeloid leukemia without

NPM1 mutations, and HOXA6–HOXA4 were pre-

dicted as targets of miR-22 [31]. miR-22 has been

detected with a distinct expression pattern in human

Duchenne muscular dystrophy, where HSPG2,

GPNMB and CLIC4 were predicted as its potential

targets [32]. HSPG2 has been reported to contribute to

tumor growth and angiogenesis in vivo [35], and

GPNMB was identified as a pathological and diagnos-

tic marker in melanocyte tumor progression [36].

Moreover, CLIC4 was found to participate in stress-

induced apoptosis in human osteosarcoma cells [37].

Flow cytometry analysis showed that overexpression

of miR-22 could reduce ERa and SP1 protein levels in

pancreatic cancer cells [33].

Estrogen receptors (mainly ERa and ERb) constitutea group of ligand-activated nuclear receptors that are

activated by estrogen. Human ERa is a transcription

factor that regulates diverse gene expression, and is

implicated in cancers by stimulating cell proliferation

and tumor growth [38,39]. An miRNA library-based

screening with miR-206, miR-18a and miR-221 ⁄ 222 as

putative positive controls [17,40,41] demonstrated that

miR-22 could robustly suppress the luciferase signal of

ERa 3¢-UTR tethered vector. By mutating the comple-

mentary site for the miR-22 seed region, we showed that

repression of ERa by miR-22 was almost completely

abolished. Conversely, the ERa signal intensities were

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.



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

GAPDH mRNA. Data are presented as mean ± standard deviation from at least three independent experiments. (B) Regulation of ERa

expression by two ERa siRNAs. MCF-7 cells were transfected with control RNA duplex or ERa siRNAs, total cellular proteins were prepared

and analyzed for ERa protein expression by western blotting at 48 h after transfection. b-actin was used as a loading control and the relative

density of bands was densitometrically quantified. (C, D) Knockdown of ERa could recapitulate the phenotype of repressed cell growth

induced by miR-22 overexpression. MCF-7 cells were transfected with control RNA duplex or miR-22 duplex or ERa siRNAs for 24 h incuba-

tion. In the other two groups, reintroduction of ERa abrogates the antigrowth effect of miR-22. MCF-7 cells were first transfected with

miR-22 duplex, and at 24 h after transfection, sequentially transfected with ERa-expressing vector pcDNA3.1–ESR1 (indicated as ERa) or

empty vector pcDNA3.1 (empty vector) for 24 h incubation. (C) Single-cell suspensions containing 20 000 cells treated respectively were

seeded in each well of 24-well plates at 37 �C as attached monolayers in DMEM containing 10% fetal bovine serum. Cells were harvested

by treatment with trypsin and counted every 24 h in triplicate. (D) Single-cell suspensions containing 10 000 cells treated respectively were

seeded in each well of six-well plates coated with soft agar. The plates were photographed after incubation at 37 �C for 2 weeks. Data are

presented as mean ± standard deviation from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, compared with

control-RNA-duplex-transfected cells or comparison between two groups as indicated.



In summary, we showed that frequent downregula-

tion of miR-22 expression is associated with ERa-posi-tive human breast cancer cells, and miR-22 can

directly regulate ERa expression. We further showed

that ERa is potentially involved in miR-22-mediated

repression of human cancer cell growth of ERa-posi-tive breast cancer cells. It would then be interesting to

explore whether miR-22 could serve as a potential

therapeutic reagent in the treatment of cancer in which

ERa plays an important role.

Materials and methods

Cell lines and cultures

Five breast carcinoma cell lines were obtained from the Cell

Resource Center of Peking Union Medical College (Beijing,

China) and maintained in our laboratory. HEK293T

(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¢-UTR–MUT. The ERa-expressing vector (pcDNA3.1–

ESR1) was created by cloning the ESR1 coding sequence

into the EcoRI and NheI sites of pcDNA3.1 (Invitrogen).

The primers used in the subcloning experiments are

included in Table S2; all the construct products were

validated by sequencing.

RNA isolation and quantitative RT-PCR detection

Total RNAs from cultured cells were isolated using TRI

Reagent (Sigma, St Louis, MO, USA) and converted into

cDNA using ImPro-II reverse transcriptase (Promega).

Detection of the mature form of miR-22 was performed

using Quantitect SYBR Green PCR Kit (Qiagen, Hilden,

Germany) and quantitative RT-PCR Primer Sets (Ribo-

bio, Guangzhou, China) with the U6 small nuclear RNA

as an internal control. Detection of the ERa mRNA was

performed using Quantitect SYBR Green PCR Kit

(Qiagen), with glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) mRNA as an internal control. The detection

primers for ESR1 and GAPDH are included in Table S2.

RNA oligoribonucleotides and cell transfections

The miRNA mimic library was obtained from Ribobio

(Guangzhou, China); miR-22 duplex and the negative con-

trol RNA duplex (indicated as control RNA duplex) were

obtained from GenePharma (Shanghai, China) (Fig. S1).

The control RNA duplex was used to eliminate the poten-

tial nonsequence-specific effects and its sequences were non-

homologous to any human genome sequences. MCF-7 cells

were transfected with 50 nm RNA duplex using lipofecta-

mine 2000 (Invitrogen) and counted 24 h after transfection

for plating wells to observe proliferation and colony forma-

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¢. Thenegative 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¢; ERasiRNA #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).



new

高亮

new

高亮

Luciferase reporter assay

For miRNA duplex library analysis, HEK293T and MCF-7

cells per well in 24-well plates were cotransfected with 50 nm

miRNA duplex, 120 ng pGL3m–ESR1–3¢-UTR–WT and

8 ng pRL-TK (Promega) in duplicate. For miR-22 analysis,

cells were cotransfected with plasmids (300 ng pcDNA3–

miR-22 or pcDNA3.0) or duplexes (33 nm miR-22 duplex or

control RNA duplex), 120 ng pGL3m–ESR1–3¢-UTR–WT

or pGL3m–ESR1–3¢-UTR–MUT and 8 ng pRL-TK in trip-

licate. In MDA-MB-231 cells, cells were cotransfected with

200 nm anti-miR-22 or control anti-miR, 120 ng pGL3m–

ESR1–3¢-UTR–WT or pGL3m–ESR1–3¢-UTR–MUT and

8 ng pRL-TK in triplicate. For the above analyses, cell

lysates were analyzed 48 h after transfection using the Dual-

Luciferase Reporter Assay System (Promega) and the experi-

ments were independently repeated at least three times.

Luciferase activity was detected using the Synergy HT micro-

plate fluorescence reader (Bio-Tek Instruments, Winooski,

VT, USA). The pRL-TK vector constitutively expressing

Renilla luciferase was cotransfected as an internal control to

minimize experimental variability caused by the differences

in cell viability or transfection efficiencies.

Tissue specimens and RNA extraction

Fresh breast cancer tissues of 23 individual patients were

procured from surgical resection specimens collected in the

Cancer Hospital, Chinese Academy of Medical Sciences

(Beijing, China) in 2009. The clinical characteristics of

patients with breast carcinoma are shown in Table 1. No

patients received treatment before surgery and they signed

informed consent forms for sample collection. ERa protein

expression status was confirmed at diagnosis using standard

immunohistochemistry procedures. Total RNA was isolated

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 · 104

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

30871385).

References

1 Bartel DP (2004) MicroRNAs: genomics, biogenesis,

mechanism, and function. Cell 116, 281–297.

2 Lu M, Zhang Q, Deng M, Miao J, Guo Y, Gao W &

Cui Q (2008) An analysis of human microRNA and

disease associations. PLoS ONE 3, e3420.

3 Esquela-Kerscher A& Slack FJ (2006) Oncomirs-microR-

NAs with a role in cancer.Nat Rev Cancer 6, 259–269.

4 Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch

E, Yendamuri S, Shimizu M, Rattan S, Bullrich F,

Negrini M et al. (2004) Human microRNA genes are

frequently located at fragile sites and genomic regions

involved in cancers. Proc Natl Acad Sci USA 101,

2999–3004.

5 Zhang L, Huang J, Yang N, Greshock J, Megraw MS,

Giannakakis A, Liang S, Naylor TL, Barchetti A, Ward

MR et al. (2006) microRNAs exhibit high frequency

genomic alterations in human cancer. Proc Natl Acad

Sci USA 103, 9136–9141.

6 Corney DC, Flesken-Nikitin A, Godwin AK, Wang W

& Nikitin AY (2007) MicroRNA-34b and MicroRNA-

34c are targets of p53 and cooperate in control of cell

Table 1. Clinical characteristics of patients with breast carcinoma

Variable

ERa positive ERa negative

n (%) n (%)

Total no. 10 (43.5) 13 (56.5)

Age

< 50 years 3 (30.0) 6 (46.2)

‡ 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)



new

高亮

proliferation and adhesion-independent growth. Cancer

Res 67, 8433–8438.

7 Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin

M, Shimizu M, Wojcik SE, Aqeilan RI, Zupo S, Dono M

et al. (2005) miR-15 and miR-16 induce apoptosis by tar-

geting BCL2. Proc Natl Acad Sci USA 102, 13944–13949.

8 Ma L, Teruya-Feldstein J & Weinberg RA (2007)

Tumour invasion and metastasis initiated by micro-

RNA-10b in breast cancer. Nature 449, 682–688.

9 Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J,

Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferran-

do AA et al. (2005) MicroRNA expression profiles clas-

sify human cancers. Nature 435, 834–838.

10 Castanotto D & Rossi JJ (2009) The promises and pit-

falls of RNA-interference-based therapeutics. Nature

457, 426–433.

11 Parkin DM, Bray F, Ferlay J & Pisani P (2005) Global

cancer statistics, 2002. CA Cancer J Clin 55, 74–108.

12 Wooster R & Weber BL (2003) Breast and ovarian can-

cer. N Engl J Med 348, 2339–2347.

13 Hanahan D & Weinberg RA (2000) The hallmarks of

cancer. Cell 100, 57–70.

14 Zhu S, Si ML, Wu H & Mo YY (2007) MicroRNA-21

targets the tumor suppressor gene tropomyosin 1

(TPM1). J Biol Chem 282, 14328–14336.

15 Iorio MV, Casalini P, Piovan C, Di Leva G, Merlo A,

Triulzi T, Menard S, Croce CM & Tagliabue E (2009)

microRNA-205 regulates HER3 in human breast can-

cer. Cancer Res 69, 2195–2200.

16 Hossain A, Kuo MT & Saunders GF (2006) Mir-17-5p

regulates breast cancer cell proliferation by inhibiting

translation of AIB1 mRNA. Mol Cell Biol 26,

8191–8201.

17 Adams BD, Furneaux H & White BA (2007) The

micro-ribonucleic acid (miRNA) miR-206 targets the

human estrogen receptor-a (ERa) and represses ERamessenger RNA and protein expression in breast cancer

cell lines. Mol Endocrinol 21, 1132–1147.

18 Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo

R, Sabbioni S, Magri E, Pedriali M, Fabbri M,

Campiglio M et al. (2005) MicroRNA gene expression

deregulation in human breast cancer. Cancer Res 65,

7065–7070.

19 Datta J, Kutay H, Nasser MW, Nuovo GJ, Wang B,

Majumder S, Liu CG, Volinia S, Croce CM, Schmitt-

gen TD et al. (2008) Methylation mediated silencing of

microRNA-1 gene and its role in hepatocellular carcino-

genesis. Cancer Res 68, 5049–5058.

20 Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP &

Burge CB (2003) Prediction of mammalian microRNA

targets. Cell 115, 787–798.

21 Rajewsky N (2006) MicroRNA target predictions in

animals. Nat Genet 38(Suppl), S8–S13.

22 Zhao Y, Samal E & Srivastava D (2005) Serum

response factor regulates a muscle-specific microRNA

that targets Hand2 during cardiogenesis. Nature 436,

214–220.

23 Kuhn DE, Martin MM, Feldman DS, Terry AV Jr,

Nuovo GJ & Elton TS (2008) Experimental validation

of miRNA targets. Methods 44, 47–54.

24 Berezikov E, Cuppen E & Plasterk RH (2006)

Approaches to microRNA discovery. Nat Genet 38

(Suppl), S2–S7.

25 Griffiths-Jones S, Saini HK, van Dongen S & Enright

AJ (2008) miRBase: tools for microRNA genomics.

Nucleic Acids Res 36, D154–D158.

26 He L & Hannon GJ (2004) MicroRNAs: small RNAs

with a big role in gene regulation. Nat Rev Genet 5,

522–531.

27 Evan GI & Vousden KH (2001) Proliferation, cell cycle

and apoptosis in cancer. Nature 411, 342–348.

28 Johnson CD, Esquela-Kerscher A, Stefani G, Byrom

M, Kelnar K, Ovcharenko D, Wilson M, Wang X,

Shelton J, Shingara J et al. (2007) The let-7 microRNA

represses cell proliferation pathways in human cells.

Cancer Res 67, 7713–7722.

29 Hayashita Y, Osada H, Tatematsu Y, Yamada H,

Yanagisawa K, Tomida S, Yatabe Y, Kawahara K,

Sekido Y & Takahashi T (2006) A polycistronic micr-

oRNA cluster, miR-17-92, is overexpressed in human

lung cancers and enhances cell proliferation. Cancer Res

65, 9628–9632.

30 Lagos-Quintana M, Rauhut R, Lendeckel W & Tuschl

T (2001) Identification of novel genes coding for small

expressed RNAs. Science 294, 853–858.

31 Garzon R, Garofalo M, Martelli MP, Briesewitz R,

Wang L, Fernandez-Cymering C, Volinia S, Liu CG,

Schnittger S, Haferlach T et al. (2008) Distinctive micr-

oRNA signature of acute myeloid leukemia bearing

cytoplasmic mutated nucleophosmin. Proc Natl Acad

Sci USA 105, 3945–3950.

32 Eisenberg I, Eran A, Nishino I, Moggio M, Lamperti

C, Amato AA, Lidov HG, Kang PB, North KN, Mitra-

ni-Rosenbaum S et al. (2007) Distinctive patterns of

microRNA expression in primary muscular disorders.

Proc Natl Acad Sci USA 104, 17016–17021.

33 Sun M, Estrov Z, Ji Y, Coombes KR, Harris DH &

Kurzrock R (2008) Curcumin (diferuloylmethane) alters

the expression of microRNAs in human pancreatic can-

cer cells. Mol Cancer Ther 7, 464–473.

34 Pandey DP & Picard D (2009) MiR-22 inhibits estrogen

signaling by directly targeting the estrogen receptor

alpha mRNA. Mol Cell Biol 29, 3783–3790.

35 Sharma B, Handler M, Eichstetter I, Whitelock JM,

Nugent MA & Iozzo RV (1998) Antisense targeting of

perlecan blocks tumor growth and angiogenesis in vivo.

J Clin Invest 102, 1599–1608.

36 Weterman MA, Ajubi N, van Dinter IM, Degen WG,

van Muijen GN, Ruitter DJ & Bloemers HP (1995)

nmb, a novel gene, is expressed in low-metastatic



human melanoma cell lines and xenografts. Int J Cancer

60, 73–81.

37 Fernandez-Salas E, Suh KS, Speransky VV, Bowers

WL, Levy JM, Adams T, Pathak KR, Edwards LE,

Hayes DD, Cheng C et al. (2002) mtCLIC ⁄CLIC4, anorganellular chloride channel protein, is increased by

DNA damage and participates in the apoptotic

response to p53. Mol Cell Biol 22, 3610–3620.

38 Deroo BJ & Korach KS (2006) Estrogen receptors and

human disease. J Clin Invest 116, 561–570.

39 Clarke RB, Anderson E & Howell A (2004) Steroid

receptors in human breast cancer. Trends Endocrinol

Metab 15, 316–323.

40 Liu WH, Yeh SH, Lu CC, Yu SL, Chen HY, Lin CY,

Chen DS & Chen PJ (2009) MicroRNA-18a prevents

estrogen receptor-alpha expression, promoting prolifera-

tion of hepatocellular carcinoma cells. Gastroenterology

136, 683–693.

41 Zhao JJ, Lin J, Yang H, Kong W, He L, Ma X,

Coppola D & Cheng JQ (2008) MicroRNA-221 ⁄ 222negatively regulates estrogen receptor alpha and is

associated with tamoxifen resistance in breast cancer.

J Biol Chem 283, 31079–31086.

Supporting information

The following supplementary material is available:

Fig. S1. The sequences of synthetic miR-22 duplex and

negative control RNA duplex.

Table S1. Partial predicted miRNAs that target ESR1

3¢-UTR by targetscan, miranda or pictar.

Table S2. Primers for cloning miR-22, ESR1 3¢-UTR

and ESR1 coding sequence and for the detection of

ESR1 and GAPDH mRNA by quantitative RT-PCR.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and

may be re-organized for online delivery, but are not

copy-edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.



An estrogen receptor α suppressor, microRNA22, is downregulated in estrogen receptor α-positive human breast cancer cell lines and clinical samples

Documents