downregulated in estrogen receptor a-positive humanbreast cancer cell lines and clinical samples Jianhua Xiong1,*, Dianke Yu2,*, Na Wei1, Hanjiang Fu3, Tianjing Cai1, Yuanyu Huang1, Chen
Trang 1downregulated in estrogen receptor a-positive human
breast cancer cell lines and clinical samples
Jianhua Xiong1,*, Dianke Yu2,*, Na Wei1, Hanjiang Fu3, Tianjing Cai1, Yuanyu Huang1, Chen Wu2, Xiaofei Zheng3, Quan Du1, Dongxin Lin2and 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: liangz@pku.edu.cn
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: lindx72@cicams.ac.cn
*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-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.
Trang 2frequencies 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) 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 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 )8.40 kcalÆmol)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-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
Trang 3form 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 ERa expression 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.
Trang 4A B
G F
E
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 concon-trol 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 exSup-pression 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.
Trang 5Frequent 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
negative (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-positive
breast 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 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, comparicompari-son 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).
Trang 6immunoblot 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
C17orf91gene [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 mutareduc-tions in NPM1compared 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) constitute
a 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
Trang 7C
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.
Trang 8In 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)1insulin
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 concon-trol 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¢ 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)
Trang 9Luciferase 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
Renillaluciferase 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)1nitroblue 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)
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