THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 38, pp. 27480 –27493, September 20, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Comprehensive Analysis of MicroRNA (miRNA) Targets in
Breast Cancer Cells*□
S
Received for publication, June 6, 2013, and in revised form, August 6, 2013 Published, JBC Papers in Press, August 6, 2013, DOI 10.1074/jbc.M113.491803
Meiyun Fan1, Raisa Krutilina, Jing Sun, Aarti Sethuraman, Chuan He Yang, Zhao-hui Wu, Junming Yue,
and Lawrence M. Pfeffer
From the Departments of Pathology and Laboratory Medicine, Center for Adult Cancer Research, University of Tennessee Health
Science Center, Memphis, Tennessee 38163
Background: miRNA deregulation contributes to tumor progression.
Results: Endogenous miRNA targets were identified in two breast cancer cell lines by integrated analysis of miRNA/mRNA
expression and miRNA-mRNA interaction.
Conclusion: miRNAs collectively function to promote survival but suppress cell migration/invasion.
Significance: The defined endogenous miRNA targets will facilitate future studies to link miRNA deregulation with breast
cancer cell properties.
* This work was supported, in whole or in part, by National Institutes of Health
Grants CA140346 (to M. F.) and CA133322 (to L. M. P.).
This article contains supplemental Table S1.
All expression microarray data were deposited in the Gene Expression Omnibus
with accession number GSE48162.
1
To whom correspondence should be addressed: 19 South Manassas St.,
Memphis, TN 38163. Tel.: 901-448-4192; Fax: 901-448-3910; E-mail:
mfan2@uthsc.edu.
□
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MicroRNAs (miRNAs)2 are emerging as key modulators of
gene expression at the post-transcriptional level by repressing
translation and/or inducing mRNA degradation (1, 2). Most
miRNAs are initially transcribed as long primary transcripts
(pri-miRNAs) that are processed within the nucleus into short
stem-loops (pre-miRNAs) by DROSHA, a member of the ribonuclease III superfamily of double-stranded RNA-specific
endoribonucleases (3). The pre-miRNAs are transported to the
cytoplasm and further processed by DICER1, another doubledstranded RNA-specific ribonuclease, to generate mature
miRNAs, which are loaded into the RNA-induced silencing
complexes (RISCs) (4). miRNAs recruit mRNA targets to
RISCs through Watson-Crick base pairing (2). Computational
sequence analysis and experimental evidence suggest that bases
2– 8 at the 5⬘-end of mature miRNAs (termed seed sequences)
and their complementary sequences located in the 3⬘-untranslated region (3⬘-UTR) of mRNA are the major determinants of
miRNA-mRNA interaction (5– 8). A single miRNA can target
hundreds of mRNAs, and a single mRNA can be coordinately
regulated by multiple miRNAs (7, 9). Approximately 60% of
mammalian mRNAs have one or more evolutionarily conserved miRNA target sequences (7). However, it is unclear
whether a miRNA exerts its effects via regulating its entire repertoire of targets or a subset of specific effectors in a given cell
context. The complexity of miRNA function can hardly be
depicted by traditional studies that focus on a single miRNA
and its predicted targets one at a time. A prerequisite for understanding the collective function of endogenous miRNAs is to
determine what mRNAs and signaling pathways are targeted by
miRNAs under physiologically relevant conditions.
Argonaute proteins are the catalytic components of the
RISCs for mRNA silencing or destruction. All four human argonaute proteins (AGO1, AGO2, AGO3, and AGO4) are able to
interact with miRNAs as components of RISCs to inhibit translation, but only AGO2 possesses the endoribonuclease activity
2
The abbreviations used are: miRNA, microRNA; pri-miRNA, primary miRNA;
IP, immunoprecipitation; AGO2-IP, AGO2 immunoprecipitation; RISC,
RNAi-induced silencing complex; KD, knockdown; Ab, antibody; qPCR,
quantitative PCR; APA, alternative polyadenylation.
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MicroRNAs (miRNAs) regulate mRNA stability and translation through the action of the RNAi-induced silencing complex.
In this study, we systematically identified endogenous miRNA
target genes by using AGO2 immunoprecipitation (AGO2-IP)
and microarray analyses in two breast cancer cell lines, MCF7
and MDA-MB-231, representing luminal and basal-like breast
cancer, respectively. The expression levels of ⬃70% of the
AGO2-IP mRNAs were increased by DROSHA or DICER1
knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3ⴕ-UTR of
mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the 50 most abundantly expressed miRNAs.
Together, these results suggested that the majority of the
AGO2-associated mRNAs were bona fide miRNA targets.
Functional enrichment analysis uncovered that the AGO2-IP
mRNAs were involved in regulation of cell cycle, apoptosis,
adhesion/migration/invasion, stress responses (e.g. DNA damage and endoplasmic reticulum stress and hypoxia), and cell-cell
communication (e.g. Notch and Ephrin signaling pathways). A
role of miRNAs in regulating cell migration/invasion and stress
response was further defined by examining the impact of DROSHA
knockdown on cell behaviors. We demonstrated that DROSHA
knockdown enhanced cell migration and invasion, whereas it sensitized cells to cell death induced by suspension culture, glucose
depletion, and unfolding protein stress. Data from an orthotopic
xenograft model showed that DROSHA knockdown resulted in
reduced growth of primary tumors but enhanced lung metastasis.
Taken together, these results suggest that miRNAs collectively
function to promote survival of tumor cells under stress but suppress cell migration/invasion in breast cancer cells.
miRNA Targets in Breast Cancer Cells
EXPERIMENTAL PROCEDURES
Cell Culture—MCF7 and MDA-MB-231 were purchased
from ATCC (Manassas, VA) and cultured in minimal essential
medium supplemented with 10% fetal bovine serum (FBS) and
100 units/ml penicillin-streptomycin. To generate cells that
stably express shRNA against DROSHA or DICER1, cells were
transduced with lentivirus containing pSicoR-Drosha1 or
pSicoR-Dicer1 (Addgene 14766 or 14763) (34) and selected in
medium supplemented with 2 g/ml puromycin.
AGO2 Immunoprecipitation—Cells (3 ⫻ 107) were suspended in 3 ml of ice-cold hypotonic buffer (10 mM Tris (pH
7.5), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 100 units/ml RNase
OUT, and protease inhibitor mixture) for 15 min. The cytoplasmic fraction was isolated by homogenization with a Dounce
homogenizer and centrifugation at 14,000 ⫻ g at 4 °C for 10 min
and incubated with control IgG (5 g of Ab/mg of lysate) and
anti-mouse IgG-coated magnetic beads for 1 h to eliminate
nonspecific binding. The precleaned lysates were then mixed
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with mouse anti-human Ago2 (5 g of Ab/mg of lysate; clone
2E12-1C9, Abnova (Taipei City, Taiwan)) and anti-mouse IgGcoated magnetic beads. After incubation overnight at 4 °C on a
rocking platform, AGO2-IP beads were washed twice with icecold wash buffer (hypotonic buffer supplemented with 150 mM
NaCl and 0.5% Nonidet P-40) and once with high salt buffer
(hypotonic buffer supplemented with 400 mM NaCl and 0.5%
Nonidet P-40). RNA and protein were extracted from the
AGO2-IP complexes using TRIzol (Invitrogen) and Laemmli
buffer, respectively.
Quantitation of mRNA, miRNA, and pri-miRNA Expression
Using qPCR—Total RNA was converted to cDNA by using
iScript cDNA synthesis kits (Bio-Rad) or the NCodeTM miRNA
First-Strand cDNA Synthesis Kit (Life Technologies) for
mRNA or miRNA detection, respectively. qPCR was performed
on the CFX96TM Real-Time PCR Detection System using SYBR
Green supermix (Bio-Rad). Expression data of mRNA and miRNA
were normalized to GAPDH and U6 snRNA, respectively, using
the 2⫺⌬⌬CT method, and presented as mean ⫾ S.E. (n ⫽ 3). qPCR
primers were obtained from PrimerBank or designed using
Primer3Plus (35, 36). The expression levels of Pri-miRNA were
examined by using TaqMan Pri-miRNA assays according to the
manufacturer’s instructions (Invitrogen).
Immunoblotting—Protein extracts were resolved in SDSPAGE, transferred to PVDF membrane, and immunoblotted
with the indicated antibodies. Antibodies for DROSHA,
MAP1LC3, and GAPDH were from Cell Signaling Technologies (Boston, MA), and AGO2 was from Abnova.
Microarray Analysis—The purified RNA samples from
whole cells (input RNA) and AGO2-IP were submitted to the
University of Tennessee Health Science Center Center of
Genomics and Bioinformatics (Memphis, TN) for labeling and
hybridization to HT-12 expression BeadChips (Illumina Inc.).
Three independent AGO2-IP experiments were performed.
Hybridization signals were processed (annotation, background
subtraction, quantile normalization, and presence call filtering)
using Illumina Genome Studio software (Illumina). AGO2-IPenriched mRNAs were identified using Genespring GX version
9.0 (Agilent Technologies Inc., Santa Clara, CA) with the following cut-offs: false discovery rate ⫽ 0.1 (AGO2-IP versus
input, n ⫽ 3), -fold enrichment (AGO2-IP versus input) ⱖ 1.5 in
more than 2 of 3 experiments. Functional annotation and pathway mapping of the AGO2-IP mRNAs were performed by Ingenuity pathway analysis (Ingenuity Systems, Inc., Redwood City,
CA). The microarray data can be found in the Gene Expression
Omnibus database with accession number of GSE48162.
Apoptosis Assays—To induce anoikis, cells (5 ⫻ 104/well)
were seeded in 6-well dishes coated with polyHEMA (Sigma) to
prevent cell attachment. To induce endoplasmic reticulum
stress, cells were treated with thapsigargin (50 nM). For glucose
depletion, cells were seeded in growth medium overnight,
washed with PBS twice, and cultured in glucose-free medium
for 16 h. The glucose-free medium consists of DMEM (without
glucose; Life Technologies), 5% dialyzed FBS (Life Technologies), and 100 units/ml penicillin-streptomycin. Apoptotic cells
with compromised membrane integrity were detected with
YO-PRO-1 dye according to the manufacturer’s instructions
(Life Technologies), followed by flow cytometer analysis.
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to catalyze small RNA-directed, site-specific mRNA cleavage
(10, 11). In addition, AGO2 is the most abundant argonaute
protein in the majority of mammalian tissues, including mammary gland (12–14). Therefore, AGO2 probably plays a key role
in RNA-induced silencing in mammary gland epithelial cells.
Several studies have demonstrated that miRNA targets can be
identified from immunopurified AGO2 complexes (6, 15–21).
Among the various approaches developed to identify miRNA
targets, AGO2 immunoprecipitation (AGO2-IP), combined
with mRNA expression microarray analysis, represents a direct
and feasible approach to systematically identify miRNA targets
in a physiologically relevant manner, which was employed in
our study to investigate miRNA targets in breast cancer cells.
Such systematic studies will advance our understanding of the
complex features of miRNA function.
Deregulation of miRNAs is associated with breast cancer
development and progression (22–33). Although several key
targets of breast cancer-associated miRNAs have been identified and linked to tumor phenotypes, the gene networks orchestrated by miRNAs in breast cancer cells are largely unknown. In
this study, we performed AGO2-IP, followed by expression
microarray analysis, to systematically identify miRNA targets in
MCF7 and MDA-MB-231, the two widely used cell lines that
represent luminal estrogen-dependent and basal-like triple
negative breast tumors, respectively. The numbers of mRNAs
detected in AGO2-IP from MCF7 and MDA-MB-231 cells were
877 and 703, respectively (false discovery rate ⫽ 0.1). In silico
analysis of the 3⬘-UTRs of these AGO2-IP mRNAs as well as
their expression in cells with impaired miRNA synthesis suggested that the majority of the AGO2-IP mRNAs were bona fide
miRNA targets. Functional enrichment analysis revealed that
the endogenous miRNAs predominantly target genes that regulate cell cycle, apoptosis, adhesion/migration/invasion, stress
responses (e.g. DNA damage, hypoxia, and endoplasmic reticulum stress), and cell-cell communication (e.g. Notch and Ephrin signaling pathways). Accordingly, inhibiting miRNA processing by DROSHA or DICER1 knockdown enhanced cell
ability for migration and invasion but sensitized cells to apoptosis induced by various types of stress.
miRNA Targets in Breast Cancer Cells
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sequences (forward, 5⬘-ACG CCTGTAATCCCAGCACTT-3⬘;
reverse, 5⬘-TCGCCCAGGCTGGAGTGCA-3⬘) (38).
Statistical Analysis—Data from two or three independent
experiments with replicates are presented as means ⫾ S.D.
Analysis of variance and post hoc least significant difference
analysis or t tests were performed using GraphPad Prism 5 software. p values of ⬍0.05 (*) were considered statistically
significant.
RESULTS
Identification of Endogenous miRNA Targets in Luminal and
Basal-like Breast Cancer Cells—MCF7 and MDA-MB-231 cells
were chosen for study because they are the most frequently
used cell lines that represent luminal and basal-like breast cancer, respectively. A better understanding of the regulatory networks of gene expression in these two cell lines is critical to
understand changes in breast cancer cell behavior elicited by
various types of stress or genetic manipulations. AGO2 is the
most abundantly expressed argonaute protein in mammary
gland (see the Tissue-specific Gene Expression and Regulation
(TiGER) Web site). Therefore, AGO2 probably plays a key role
in RNA-induced silencing in mammary gland epithelial cells,
and mRNAs coimmunoprecipitated with AGO2 may well represent the majority of endogenous miRNA targets.
First we examined the specificity and AGO2-IP efficiency of a
mouse monoclonal AGO2 antibody (clone 2E12-1C9, Abnova).
When whole cell lysates were used for immunoblotting, the
antibody recognized a single band at ⬃95 kDa in both MCF7
and MDA-MB-231 cells (Fig. 1A, top). The efficiency of the
AGO2 antibody for IP was confirmed by enrichment of AGO2
protein in the IP complexes (anti-AGO2 versus control IgG)
and depletion of AGO2 protein in the IP flow-through (Fig. 1A,
bottom). Next, we examined the enrichment of miRNA targets
in the AGO2-IP complexes. As shown in Fig. 1B, several
mRNAs that have been established as miRNA targets in
MCF7 cells (i.e. BTG2, CCNE1, CDC25A, DICER1, EZH2, and
RUNX1) were significantly enriched by AGO2-IP (p ⬍ 0.05,
AGO2-IP versus IgG-IP).
To systematically identify mRNA targets, total RNA was isolated from AGO2-IP complexes and subjected to microarray
analysis using human HT-12 expression BeadChips (Illumina
Inc.). Three independent IP and array analysis were conducted.
Using a cut-off set that combined false discovery rate and -fold
enrichment (false discovery rate ⫽ 0.1 (AGO2-IP versus input,
n ⫽ 3), -fold enrichment (AGO2-IP versus input) ⱖ 1.5 in more
than 2 of 3 independent biological repeats), 877 and 703
mRNAs were detected in AGO2-IP from MCF7 in MDA-MB231, respectively (Fig. 1C and supplemental Table S1 and
GSE48162). The AGO2-IP mRNAs from the two cell lines
shared a marked overlap and also exhibited cell type-specific
mRNA-AGO2 interaction, as summarized in Fig. 1C. The differences between these two cell lines may reflect the differential
expression of mRNAs and miRNAs as well as the presence of
different isoforms of mRNAs due to alternative splicing and/or
polyadenylation.
Identification of Signaling Pathways Targeted by miRNAs—
To understand the physiological role of these miRNA targets,
we performed functional enrichment analysis of Ago2-IP
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Transient Transfection and Luciferase Reporter Assay—
CMV-d2eGFP-21 (miR-21 sponge), CMV-d2eGFP-empty
(vector control for miR-21 sponge) and pCMV-luc-miR21
(luciferase reporter with miR-21 target sites have been characterized previously (Addgene 21927, 26164, and 20876) (37). To
examine the efficiency of the miR-21 sponge to inhibit miR-21
function, MDA-MB-231 cells were transfected with pCMVluc-miR21, along with CMV--galactosidase and various doses
of CMV-d2eGFP-21 or vector control, using Lipofectamine
2000 (Life Technologies). Luciferase and -galactosidase activities were measured 48 h after transfection using the luciferase
and -galactosidase assay system, respectively (Promega, Madison, WI). Luciferase activity was normalized to -galactosidase
activity and expressed as mean ⫾ S.E. (n ⫽ 6). To examine the
effect of miR-21 inhibition on interaction between AGO2 and
miR-21 targets, MDA-MB-231 cells (1 ⫻ 107) were transfected
with 15 g of CMV-d2eGFP-21 or empty vector using Lipofectamine 2000, followed by AGO2-IP 48 h after transfection.
RNA samples prepared from whole cells and AGO2-IP were subjected to qPCR analysis. To examine the effect of miRNA inhibition on mRNA expression, cells (4 ⫻ 105) were transfected with 50
nM miRCURY LNA miRNA inhibitor (Exiqon) or a control oligonucleotide using Lipofectamine RNAiMAX (Invitrogen). Total
RNA was prepared 48 h after transfection and subjected to qPCR
analysis. The sequences of the miRNA inhibitors for miR-221 and
miR-200a are AACCCAGCAGACAATGTAGC and CATCGTTACCAGACAGTGTT, respectively.
Migration and Invasion Assays—Cells (20,000 cells/0.5
ml/well) were plated onto control membrane inserts with 8-m
pores or Matrigel-coated membrane inserts (BD Biosciences),
which are placed in 24-well chambers filled with 0.6 ml of
growth medium. Twenty-four hours after plating, cells that
remained on the upper surface of the membrane were removed
by cotton-tipped swabs, and cells that migrated/invaded to the
lower surface of the membrane were fixed with methanol,
stained with 0.5% crystal violet, and counted under a microscope. The percent invasion was expressed as follows: % invasion ⫽ (mean number of cells invading through Matrigel insert
membrane ⫻ 100)/mean number of cells migrating through
control insert membrane.
Orthotopic Xenograft Model and Lung Metastasis—All animal studies adhered to protocols approved by the Institutional
Animal Care and Use Committee of the University of Tennessee Health Science Center. Cells (7.5 ⫻ 105 cells in 10 l of PBS)
were surgically inoculated into the right inguinal mammary
gland fat pads of 4-week-old female NSG mice (NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ, The Jackson Laboratory). Mice were
inspected weekly for tumor appearance by visual observation
and palpation. Primary tumor outgrowth was monitored twice
a week using digital calipers. Tumor volume was calculated as
follows: volume ⫽ (width2 ⫻ length)/2. Tumor and lung tissues
were extracted 7 weeks after inoculation. The left lung lobes
were fixed with 4% paraformaldehyde and subjected to tissue
section (10 M) and H&E staining. Genomic DNA from lung
tissues (⬃20 mg from the right lung lobes) was prepared using the
Wizard Genomic DNA Purification Kit (Promega) and subjected
to qPCR analysis using primers specific for the human Alu
miRNA Targets in Breast Cancer Cells
mRNAs by using the Ingenuity pathway analysis system (Ingenuity Systems, Inc.). The signaling pathways and cellular functions commonly regulated by miRNAs in both MCF7 and
MDA-MB-231 cells included cell cycle, apoptosis, adhesion/
migration/invasion, lipid metabolism, stress response (e.g.
ATM, autophagy, endoplasmic reticulum stress, hypoxia, and
mitochondrial dysfunction), and transmembrane receptor signaling (e.g. notch, ephrin, and tumor necrosis factor) (Fig. 1C,
right). Several signaling pathways critical for luminal phenotype
of breast cancer were found to be targeted by miRNAs in MCF7,
including nuclear receptor (e.g. signaling pathways mediated by
estrogen, androgen, and retinoic acid receptor receptors),
HER-2, and p53 signaling pathways. In contrast, the Wnt/catenin signaling pathway that confers phenotypic plasticity to
basal-like breast cancer cells was targeted by miRNAs in MDAMB-231 cells (39). These results provide an overview of signaling pathways targeted by miRNA in luminal and basal-like
breast cancer cells, suggesting that miRNAs play an important
role in regulating cell response to extracellular stimuli and
transmembrane receptor-mediated cell-cell communications.
Validation of AGO2-IP mRNAs as Bona Fide miRNA Targets—
To validate that the identified AGO2-mRNA interactions were
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indeed mediated by miRNAs, we examined the effect of miR-21
inhibition on mRNA-AGO2 interaction in MDA-MB-231 cells.
A construct (CMV-d2eGFP-21) that expresses a sponge RNA
with multiple target sites complementary to miR-21 was used
to inhibit miR-21 function (37). The efficiency of miR-21
sponge to inhibit miR-21 activity was monitored by expression
of a luciferase indicator (pCMV-luc-miR21) that harbors four
copies of miR-21 target sites in the 3⬘-UTR (40). In transiently
transfected MDA-MB-231 cells, miR-21 sponge increased the
expression of the luciferase indicator in a dose-dependent manner (Fig. 2A) but showed no significant effect on the expression
of a control luciferase reporter (data not shown). Next, we
examined the effect of miR-21 sponge on AGO2 interaction of
a panel of established miR-21 targets. As shown in Fig. 2B,
miR-21 sponge significantly decreased the amount of miR-21
targets detected in AGO2-IP from MDA-MB-231 cells, including BTG2, COL4A1, DCUN1D3, EIF4EBP2, EPHA4, JAG1,
SPRY4, and ZCCHC3.
In addition, we examined the effect of miRNA inhibition on
the expression of cell type-specific AGO2-IP mRNAs by using
LNA-modified antisense oligonucleotides for miR-221 and
miR-200a, which represent cell line-specific miRNAs that are
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FIGURE 1. Identification of mRNAs associated with AGO2 in MCF7 and MDA-MB-231 cells. A, specificity and efficiency of anti-AGO2 antibody. AGO2
protein from whole cell lysate, AGO2-IP, and IP flow-through was detected using immunoblotting. GAPDH was used as loading control. B, enrichment of miRNA
targeted mRNAs by AGO2-IP. Total RNA was prepared from cell lysate or AGO2-IP and subjected to qPCR analysis. The mRNA levels were normalized to GAPDH.
The -fold enrichment was calculated with the equation, -fold enrichment ⫽ mRNA level detected in AGO2-IP/mRNA level in cell lysate, and is presented as
mean ⫾ S.E. (error bars) (n ⫽ 3). C, Venn diagrams and enriched cell signaling pathways of AGO2-IP mRNAs in MCF7 and MDA-MB-231 cells.
miRNA Targets in Breast Cancer Cells
highly expressed in MDA-MB-231 and MCF7, respectively. As
shown in Fig. 2C, miR-221 inhibition in MDA-MB-231 cells
increased the expression of a panel of miR-221 targets that were
specifically detected in AGO2-IP from MDA-MB-231 cells.
The expression of these mRNAs was not significantly affected
by anti-miR-221 in MCF7 cells (data not shown). Conversely,
miR-200a inhibition in MCF7 cells increased the expression of
a panel of miR-200a targets that were specifically found in
AGO2-IP from MCF7 (Fig. 2D). The expression of these genes
was not significantly affected by miR-200a inhibition in MDAMB-231 (data not shown). Collectively, these results suggest
that the AGO2-IP mRNAs are likely targets of endogenous
miRNAs.
DROSHA Knockdown Increases Expression of AGO2-IP
mRNAs—mRNA destabilization is closely correlated with
translation suppression by miRNAs (1). Therefore, we speculated that blocking DROSHA-mediated miRNA synthesis
would result in the accumulation of AGO2-IP mRNAs if
they are bona fide miRNA targets. To knockdown DROSHA,
cells were stably transduced with a lentiviral construct
(pSicoR-Drosha1) that expresses DROSHA-specific shRNA
(34). Immunoblotting and qPCR showed that DROSHA expression was reduced by ⬃80% at both the mRNA and protein level in
MDA-MB-231 cells expressing the shRNA (designated as
DROSHA-KD) compared with control cells that were transduced
with a lentiviral construct expressing scramble RNA (designed as
MDA-MB-231/C) (Fig. 3A). Because MCF7 showed modest
DROSHA knockdown efficiency (⬃50%), the following studies
were conducted in MDA-MB-231 only.
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To examine the effect of DROSHA knockdown on miRNA
procession, the expression levels of 13 pri-miRNAs were examined using the TaqMan Pri-miRNA assays (Invitrogen), including pri-MIRLET7D, MIR7–3HG, MIR17HG, MIR25, MIR21,
MIR22HG, MIR30B, MIR30C2, MIR100HG, MIR106A,
MIR125B2, MIR130A, and MIR221). DROSHA knockdown
significantly increased the abundance of seven pri-miRNAs,
concomitant with a decreased expression of the corresponding
mature miRNAs (Fig. 3, B and C). These results demonstrated
that DROSHA knockdown abolished processing of some, but
not all, of the pri-miRNAs in MDA-MB-231 cells. The various
effects of DROSHA knockdown on different pri-miRNAs are
probably due to the presence of multiple miRNA processing
pathways (41). This finding suggests that DROSHA knockdown
could have cell context-dependent effects, dependent on the
expression profiles of pri-miRNAs and activities of various
miRNA processing pathways.
Having demonstrated that DROSHA knockdown reduced
the expression of a subset of miRNAs, we examined its impact
on the expression of AGO2-IP mRNAs by microarray analysis
using the human HT-12 expression BeadChips. As shown in
Fig. 4, the expression levels of the vast majority of AGO2-IP
mRNAs (⬎70%) were increased by DROSHA knockdown. Similarly, the specific increase in expression levels of AGO2-IP
mRNAs was also observed in MDA-MB-231 cells with DICER1
knockdown (data not shown and GSE48162). Taken together,
these results support the conclusion that the AGO2-IP mRNAs
are bona fide miRNA targets.
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FIGURE 2. miR-21 sponge inhibits AGO2 association of miR-21 targets in MDA-MB-231 cells. A, a luciferase reporter that harbors miR-21 target sites in the
3⬘-UTR was used to monitor the efficiency of miR-21 inhibition by sponge mRNA. B, enrichment of mRNAs by AGO2-IP in the absence (black bars) and presence
(gray bars) of miR-21 sponge. The results are presented as mean ⫾ S.E. (error bars) (n ⫽ 3). *, p ⬍ 0.05 (Student’s t test). C, increased expression of miR-221 targets
in MDA-MB-231 cells transfected with miRCURY LNA miR-221 inhibitor. D, increased expression of miR-200a targets in MCF7 cells transfected with miRCURY
LNA miR-200a inhibitor. The results were presented as mean -fold change (miRNA inhibitor versus control oligonucleotide) ⫾ S.E. (n ⫽ 3). EV, empty vector.
miRNA Targets in Breast Cancer Cells
FIGURE 4. DROSHA knockdown in MDA-MB-231 cells increases expression levels of putative miRNA targets identified by AGO2-IP. A, -fold change of individual mRNAs in response to DROSHA knockdown. Gene expression was examined by array analysis using Illumina HT-12 expression BeadChips. B, box plots of expression levels of putative miRNA targets and non-miRNA targets. The box shows the 25th to 75th percentile with a line at the median. C, control. Error bars, S.D.
The Majority of AGO2-IP mRNAs Are Putative Targets of
Abundantly Expressed miRNAs—Next, we examined the relationship between AGO2-IP mRNAs and putative targets of
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miRNAs that are abundantly expressed in MCF7 and MDAMB-231 cells. miRNA expression in MCF7 and MDA-MB-231
cells has been extensively studied, and several global miRNA
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FIGURE 3. DROSHA knockdown in MDA-MB-231 cells leads to pri-miRNA accumulation and mature mRNA reduction. A, DROSHA expression levels in cells
stably expressing shRNA (MDA-MB-231/DROSHA-KD) or scramble RNA (MDA-MB-231/C). DROSHA mRNA and protein levels were examined by qPCR and immunoblotting, respectively. B, -fold changes of pri-miRNAs in response to DROSHA knockdown. Pri-miRNA levels were examined by using TaqMan pri-miRNA assays. The
results are presented as mean -fold change (DROSHA-KD versus control) ⫾ S.E. (error bars) (n ⫽ 3). *, p ⬍ 0.05 (Student’s t test). C, expression levels of mature miRNA in
DROSHA knockdown and control (C) cells. The results are presented as mean expression levels of miRNAs (normalized to U6) ⫾ S.E. (n ⫽ 3).
miRNA Targets in Breast Cancer Cells
expression data sets are publicly available (see the ArrayExpress, Gene Expression Omnibus, and the Cell Catalogue of
Somatic Mutations in Cancer web sites). The reported expression levels of individual miRNAs appear to vary greatly among
these data sets, presumably due differences in RNA sample
processing and the platforms used for miRNA profiling. To
compile a reliable list of abundantly expressed miRNAs in these
two cell lines, we performed a meta-analysis of a total of seven
data sets, as indicated in Fig. 5A. The expression levels of individual miRNAs in each data set were ranked according to Z
scores (42), and the average Z scores of seven data sets were
used to identify the 50 most abundantly expressed miRNAs in
each cell line. The relative expression levels of these miRNAs
were presented in Fig. 5A, among which 35 miRNAs exhibited
comparable expression levels in both cell lines (Group C),
whereas 28 miRNAs were differentially expressed (Groups A
and B).
27486 JOURNAL OF BIOLOGICAL CHEMISTRY
Among the abundantly expressed miRNAs grouped in Fig.
5A, ⬃50% of the miRNAs in each group were randomly chosen
to examine their association with AGO2 in MCF7 and MDAMB-231 cells. As shown in Fig. 5B, 27 miRNAs were detected in
AGO2-IP. The relative abundance of the 27 miRNAs detected
in AGO2-IP from MCF7 and MDA-MB-231 cells was consistent with the result from meta-analysis of their overall expression levels. For example, higher levels of Group A miRNAs were
detected in AGO2-IP from MCF7 than in that from MDA-MB231, whereas higher levels of Group B miRNAs were detected in
AGO2-IP from MDA-MB-231 than in that from MCF7. This
result suggests that AGO2 binding is well correlated with the
expression levels of most miRNAs.
These abundantly expressed miRNAs in MCF7 (Groups A
and C in Fig. 5A) and MDA-MB-231 (Groups B and C in Fig.
5A) harbor a total of 35 different seed sequences (Table 1). The
putative targets of these miRNAs were identified by using the
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FIGURE 5. Identification of the 50 most abundantly expressed miRNAs in MCF7 and MDA-MB-231 cells. A, heat map of miRNAs abundantly expressed in
MCF7 and MDA-MB-231 cells. The relative expression levels miRNA were calculated according to Z scores from seven publicly available data sets. B, relative
miRNA levels detected in AGO2-IP from MCF7 and MDA-MB-231 cells. The results are presented as mean ⫾ S.E. (error bars) (n ⫽ 3).
miRNA Targets in Breast Cancer Cells
TABLE 1
Seed sequences of abundantly expressed miRNAs
Seed sequence
AAAGCUG
AAAGUGC
AACAGUC
ACAUUCA
ACCCGUA
AGCAGCA
AGCUUAU
AGUGCAA
AUUGCAC
CCCUGAG
CGUACCG
GAGGGGC
GAGGUAG
GCAGCAU
GGCUCAG
GUAAACA
has-miR-320a
hsa-miR-17-5p/20b-5p/93-5p/106a-5p/
106b-5p
hsa-miR-212-5p
hsa-miR-181a-5p/181b-5p
hsa-miR-99b-5p/100-5p
hsa-miR-15b-5p/15b-5p/16-5p/195-5p
hsa-miR-21-5p
hsa-miR-130a-3p/301a-3p
hsa-miR-25-3p/92a-3p
hsa-miR-125a-5p/125b-5p
hsa-miR-126-3p
hsa-miR-423-5p
hsa-let-7a-5p/7b-5p/7c/7d-5p/
7e-5p/7i-5p
hsa-miR-103a-3p/107
hsa-miR-24-3p
hsa-miR-30a-5p/30b-5p/30c-5p/
30d-5p
hsa-miR-26a-5p
hsa-miR-27a-5p
hsa-miR-23a-3p/23b-3p
hsa-miR-10a-5p
Hsa-miR-29a-3p/29b-3p/29c-3p
hsa-miR-22-3p
hsa-miR-146a-5p/146b-5p
hsa-miR-221-3p/222-3p
hsa-miR-138-5p
hsa-miR-141-3p/200a-3p
hsa-miR-191-5p
hsa-miR-200b-3p/200c-3p/429
hsa-miR-196a-5p
hsa-miR-7-5p
hsa-miR-342-5p
hsa-miR-376a-3p
hsa-mir-203a
Hsa-miR-182
hsa-miR-96-5p
Cell line
specificicity
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
MDA-MB-231
MDA-MB-231
MDA-MB-231
MDA-MB-231
MDA-MB-231
MDA-MB-231
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
miRNA Target Filter of the Ingenuity pathway analysis system
based on 3⬘-UTR sequences of mRNAs. As summarized in
Table 2, over 60% of AGO2-IP mRNAs were predicted targets
of the abundantly expressed miRNAs in corresponding cells.
Compared with all expressed genes detected in the input RNA
samples, the predicted miRNA targets were significantly overrepresented in AGO2-IP mRNAs (p ⬍ 0.05, 2 test with Yates
correction). These results suggest that the majority of the
AGO2-IP mRNAs are targets of miRNAs abundantly expressed
in MCF7 and MDA-MB-231 cells. The absence of miRNA
target sites in the 3⬘-UTRs of ⬃40% AGO2-IP mRNAs could
be attributed to the presence of miRNA target sites outside
the 3⬘-UTRs, AGO2-mRNA interaction mediated by
miRNAs expressed at low levels, and undefined miRNA
binding sequences.
Alternative Polyadenylation Contributes to Cell Type-specific
AGO2 Interaction of mRNAs—Among the cell type-specific
AGO2-IP mRNAs are putative targets of miRNAs commonly
expressed in both MCF7 and MDA-MB-231 cells. One possible
cause of this cell type-specific AGO2 binding is differential
expression of mRNA isoforms with 3⬘-UTRs of varying lengths
(43). A previous study reported that about one-third of mRNAs
in various human tumor cells use alternative polyadenylation
(APA) to generate multiple mRNA isoforms that differ in their
3⬘-UTRs (44). The usage of APA appears to be cell context-dependent, resulting in expression of cell type-specific mRNA isoforms (44 – 47).
SEPTEMBER 20, 2013 • VOLUME 288 • NUMBER 38
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UCAAGUA
UCACAGU
UCACAUU
ACCCUGU
AGCACCA
AGCUGCC
GAGAACU
GCUACAU
GCUGGUG
AACACUG
AACGGAA
AAUACUG
AGGUAGU
GGAAGAC
GGGGUGC
UCAUAGA
UGAAAUG
UUGGCAA
UUGGCAC
MiRBase ID
To investigate whether APA plays a role in cell type-specific
mRNA-miRNA interactions in breast cancer cells, we examined the expression ratio of the extended 3⬘-UTR regions
(between the proximal and distal polyadenylation site) relative
to the coding region of a panel of mRNAs. The cell type-specific
AGO2-interacting mRNAs selected for this study are putative
targets of miRNAs commonly expressed in both MCF7 and
MDA-MB-231 cells and harbor APA sites according to the
AREsite and xPAD Expression & Poly(A) Database (44, 48). As
shown in Fig. 6, PTPRK, a MCF7-specific AGO2-IP mRNA,
showed a higher ratio of the extended 3⬘-UTR relative to coding
region in MCF7 than in MDA-MB-231 cells. This result suggests that an RTPRK isoform with a long 3⬘-UTR is preferentially produced in MCF7 cells. Conversely, PMAIP1 and
MAPK6, two mRNAs that were detected in AGO2-IP specifically in MDA-MB-231 cells, exhibited a higher ratio of their
extended 3⬘-UTRs relative to coding regions in MDA-MB-231
than in MCF7 cells. In addition, we also detected a preferential
expression of the extended 3⬘-UTRs of SIAH1, SLC35A1,
SPRY4, UBE2N, and APITD1 in MBA-MB-231 cells, where
they were found to be associated with AGO2. However, 13
mRNAs (from a total of 21 examined) showed no difference
in the expression of their extended 3⬘-UTRs, despite their
differential interaction with AGO2, which included CCDC25,
CLASP1, DGCR8, FXR1, MMD, PRKRA, RUNX1, TARBP2,
TFDP1, TRAM2, UBE2N, XPO5, and ZCCHC11. These results
suggest that APA accounts for the cell type-specific miRNA
interaction of some, but not all, mRNAs.
DROSHA Knockdown Increases Cell Migration and Invasion—
Because genes encoding proteins involved in adhesion, migration, and invasion (Fig. 1C) were overrepresented in AGO2-IP
mRNAs, we speculated that blocking miRNA processing may
alter cell migration and invasion. Boyden chamber migration
and invasion assays showed that DROSHA knockdown significantly enhanced migration and invasion of MDA-MB-231 cells
(Fig. 7A). These observations suggest that the endogenous
miRNAs of MDA-MB-231 function collectively to suppress
migration and invasion.
DROSHA Knockdown Promotes Cell Death in Response to
Various Types of Stress—Genes involved in various stress
signaling pathways were significantly enriched in AGO2-IP
mRNAs in both MCF7 and MDA-MB-231 cells, implicating a
role of miRNAs in cell damage control and adaptation. Intriguingly, among the AGO2-IP mRNAs are several critical
components of the autophagy pathway (e.g. CTSL1, DDIT4,
ERN1, HSPA5, IDUA, LAMTOR1, RAC3, ULK3, and VTI1B).
Autophagy is a catabolic process that delivers cellular components through double-membrane vesicles (autophagosomes) to
lysosomes for degradation. Autophagy plays an important role
in eliminating damaged cellular components and recycling cellular materials for macromolecular and organelle biosynthesis
and nutrient and energy homeostasis (49). Given the prominent
cytoprotective roles of autophagy, we hypothesized that blocking miRNA processing may alter autophagy activity and consequently cell sensitivity to stress.
First, we examined the effect of DROSHA knockdown on
autophagy activity by measuring the conversion of MAP1LC3A
from cytosolic (LC3A-I) to membrane-bound lipidated form
miRNA Targets in Breast Cancer Cells
TABLE 2
Distribution of miRNA targets
(LC3A-II), which correlates with autophagosome formation
(50). The increase of LC3A-II could result from two opposing
events, accelerated production of autophagosomes due to
autophagy pathway activation or blockage of autophagosome
degradation due to lysosome dysfunction. These two events can
be distinguished by examining the impact of autophagy activation (e.g. nutrient starvation) and lysosome inhibition (e.g. chloroquine treatment) on LC3A-II levels. Lysosome inhibition will
lead to LC3A-II accumulation in cells with base-line autophagic
activity. As shown in Fig. 7B, LC3A-II was not detected in
untreated control cells (MDA-MB-231/C), but it was increased
by glucose depletion or chloroquine treatment. In contrast, the
DROSHA-KD cells exhibited a higher base-line level of LC3AII, which was not affected by glucose depletion or chloroquine
treatment (Fig. 7B). These results suggest that DROSHA
knockdown impaired autophagy flux, supporting a role of
miRNAs in regulating autophagy.
Next, we examined the effect of DROSHA knockdown on cell
sensitivity to stressors that are known to activate autophagy,
including suspension culture (anoikis), glucose depletion, and
endoplasmic reticulum stress induced by thapsigargin (49,
51–53). Apoptotic cells were detected by using YO-PRO-1
staining (Invitrogen), which labels cells with compromised
membrane permeability. As shown in Fig. 7C, DROSHA knockdown significantly sensitized cells to apoptosis induced by
anoikis, endoplasmic reticulum stress, and glucose depletion.
Together, these results suggest that miRNAs play a protective
role in tumor cells under stress by regulating autophagy flux.
27488 JOURNAL OF BIOLOGICAL CHEMISTRY
DROSHA Knockdown Reduced Growth of Primary Tumors
but Enhanced Spontaneous Lung Metastasis in an Orthotopic
Xenograft Model—During initiation and the continuous expansion of solid tumors, cells are subjected to various types of stress
that activate autophagy, including hypoxia, nutrient deprivation, and alteration of extracellular matrix. In order to further
characterize the protective role of miRNAs in cells under stress,
we examined the effect of DROSHA knockdown on tumor
growth in vivo. Control and DROSHA knockdown MDA-MB231 cells (7.5 ⫻ 105 cells in PBS) were surgically inoculated into
the fourth inguinal mammary gland fat pads of 4-week-old
female NSG (NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ) mice. All
animals developed palpable tumors within 2 weeks after inoculation. However, a slower growth rate of tumors derived from
DROSHA-KD cells was observed (Fig. 8A, top). The difference
in tumor growth was further confirmed by tumor weights at 7
weeks after inoculation (Fig. 8A, bottom). H&E staining of tissue sections revealed the presence of necrotic loci in tumors
derived from DROSHA-KD cells but not in tumors from control cells (Fig. 8B). This result, together with the observation
that DROSHA-KD increased cell apoptosis in response to various
types of stress, suggests that DROSHA-dependent miRNAs support tumor cell survival.
To investigate the effect of DROSHA knockdown on metastatic potential, we examined the presence of tumor cells
in lung sections from mice 7 week after inoculation of
DROSHA-KD or control cells in the mammary gland fat pads.
As shown in Fig. 8C, small lung metastases were observed in
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*, 2 test with Yates correction, one-tailed.
**, Ref. 6, and 15–21.
miRNA Targets in Breast Cancer Cells
mice inoculated with control cells (bottom left). In contrast,
large areas of lung parenchyma were replaced by tumor cells in
mice received DROSHA-KD cells (bottom right). We further
quantified metastatic burden in the lungs by qPCR using
primers specific to human Alu sequences (38). DROSHA-KD
increased the amount of tumor cells in lungs by ⬃6-fold (Fig.
8C). These results implicate a role of DROSHA-mediated
miRNA synthesis in suppressing tumor metastasis.
DISCUSSION
Cancer-related miRNAs have emerged as promising therapeutic targets and intervention tools. However, a comprehensive understanding of cellular signaling pathways regulated by
miRNAs, which depends on identifying miRNA targets under
biologically relevant conditions, is greatly needed. In this study,
we examined endogenous miRNA targets in breast cancer cells
by an integrated analysis of AGO2-mRNA interaction, miRNA
expression, gene expression, and cell behavior changes in
response to inhibition of miRNA processing. To our knowledge, this is the first study aimed to systematically identify
miRNA targets in MCF7 and MDA-MB-231, two widely used
breast cancer cell lines that represent luminal and basal-like
breast cancer, respectively.
The AGO2-IP mRNAs identified in this study probably represent bona fide miRNA targets based on the following findings:
1) ⬃70% of AGO2-associated mRNAs exhibited increased
expression in response to inhibition of miRNA processing by
DROSHA or DICER1 knockdown, suggesting that the majority
SEPTEMBER 20, 2013 • VOLUME 288 • NUMBER 38
of AGO2-IP mRNAs were targeted by endogenous miRNAs; 2)
in silico analysis revealed that putative targets of the 50 most
abundantly expressed miRNAs were significantly overrepresented by AGO2-IP mRNAs, implicating a role of these
miRNAs in mediating AGO2-mRNA association; 3) ⬃30% of
the AGO2-IP mRNAs were previously identified as miRNA targets in cells of various origins (6, 15–21); 4) most of the signaling pathways that were overrepresented in AGO2-IP mRNAs
have been reported to be regulated by miRNAs, such as cell
cycle control, apoptosis, and adhesion/migration/invasion; and
5) we experimentally confirmed that a subset of the signaling
pathways overrepresented in AGO2-IP mRNAs were significantly affected by DROSHA knockdown. However, one limitation of our experimental approach is that it preferentially
detects mRNAs that stably bound to AGO2 and have intact
poly(A) tails, which may be biased against mRNAs that are targeted by miRNAs for rapid deadenylation and degradation.
Signaling pathway and function mapping of the AGO2-IP
mRNAs revealed that miRNAs predominantly target genes that
regulate cell cycle, apoptosis, autophagy, adhesion/migration/
invasion, membrane receptor-mediated cell-cell communication (e.g. Ephrin and Notch signaling pathways), and stress
responses (e.g. DNA damage, endoplasmic reticulum stress,
hypoxia, and mitochondria dysfunction) in both luminal and
basal-like breast cancer cells. Regulation of cell cycle and apoptosis by miRNAs has been well documented (54 – 60). Notably,
more antiproliferation and proapoptotic genes were identified
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FIGURE 6. Alternative polyadenylation contributes to cell type-specific AGO2-interaction of mRNAs. The left panel shows the presence of alternative
polyadenylation sites (red arrows) in the 3⬘-UTRs of mRNAs. The qPCR primers used to detect the expression of the extended 3⬘-UTR regions (between the
proximal and distal polyadenylation site) are showed as purple bars. The middle panel shows the cell type-specific interaction with AGO2 of the indicated mRNA.
The right panel exhibits the expression ratio of the extended 3⬘-UTR region relative to the coding region of mRNAs. Error bars, S.D.
miRNA Targets in Breast Cancer Cells
as miRNA targets than proproliferation and antiapoptotic
genes, indicating that miRNAs, in general, support cell proliferation and protect cells against apoptosis. Consistent with this
hypothesis, global miRNA elevation due to increased activity of
XPO5 was found to be critical for cell G1/S entry, whereas
global miRNA inhibition by DROSHA knockdown in human
colon adenocarcinoma HT29 cells has been shown to enhance
apoptosis induced by 5-fluorouracil treatment (61, 62).
miRNA deregulation has been frequently described in metastatic tumors, implicating a role of miRNAs in regulating cell
properties associated with metastasis (63). We found that genes
involved in cell adhesion/migration/invasion were overrepresented in AGO2-IP mRNAs, and DROSHA knockdown significantly enhanced cell migration and invasion in vitro and
enhanced spontaneous lung metastasis in an orthotopic xenograft model. These results suggest that miRNAs collectively
function to inhibit cell migration and invasion, which is consistent with the observation that miRNA down-regulation
rather than up-regulation occurs frequently in metastatic
tumor cells (63). In support, a recent high throughput study
showed that over 20% of the 904 human miRNAs have regula-
27490 JOURNAL OF BIOLOGICAL CHEMISTRY
tory activity on migration and invasion of cancer cells from
diverse origins, and most of these miRNAs exhibited suppressive impact (60). In addition, DICER1 down-regulation has
been shown to enhance tumor metastasis (64).
One intriguing finding of our study is that a large number of
genes critically involved in cell stress response were miRNA
targets. In solid tumors, cells must adapt continuously to
fluctuations in their microenvironment, including hypoxia,
nutrient deprivation, therapeutic insults, and alteration of
extracellular signals (e.g. extracellular matrix, cytokines, and
hormones). Cell response to environment changes involves
concerted action of diverse signaling pathways to eliminate
damage and facilitate adaptation. Recent studies suggest that
autophagy is a common downstream event of various types of
cellular stress and plays an important role in promoting tumor
cell survival and adaptation (49, 51–53, 65, 66). Autophagy is a
catabolic process that delivers cellular components through
double-membrane vesicles (autophagosomes) to lysosomes for
degradation, allowing cells to eliminate damaged components
and recycle cellular materials for macromolecular and organelle biosynthesis and nutrient and energy homeostasis (49). We
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FIGURE 7. Global miRNA inhibition by DROSHA knockdown in MDA-MB-231 cells enhances cell migration and invasion, but promotes cell death in
response to various types of stress. A, DROSHA knockdown increases cell potential for migration and invasion, which were detected by Boyden chamber
assays with uncoated or Matrigel-coated membrane, respectively. The results are presented as mean number of cells/field ⫾ S.E. (error bars) (n ⫽ 3). C, control.
B, DROSHA knockdown impairs autophagy flux, indicated by the lack of response of MAP1LC3A to glucose depletion (GD) or chloroquine (CQ) treatment. In
control cells with normal autophagy activity, glucose depletion induces conversion of MAP1LC3A from cytosolic (LC3A-I) to membrane-bound lipidated form
(LC3A-II) due to increased autophagosome assembly, whereas chloroquine causes accumulation of LC3A-II by inhibiting autophagosome degradation by
lysosome. C, DROSHA knockdown sensitizes cells to apoptosis induced by various types of stress. Apoptotic cells with compromised membrane integrity were
detected with YO-PRO-1 dye, followed by flow cytometer analysis.
miRNA Targets in Breast Cancer Cells
found that blocking DROSHA-mediated miRNA synthesis led
to impaired autophagy flux and sensitized cells to apoptosis
induced by various stressors that activate autophagy. Our data
from in vivo studies provided further evidence supporting a
protective role of miRNAs against cell death. Our results suggest that miRNAs collectively function to maintain proper
autophagy flux and protect cells against stress-induced cell
death. Given the critical roles of miRNA and autophagy in cell
homeostasis, the interaction between these two pathways warrants further investigation.
Cell type-specific effects of miRNAs have been recognized,
but the underlying mechanism is not clear. One potential
mechanism is the presence of mRNA isoforms with various
lengths of 3⬘-UTRs due to the usage of alternative polyadenylation sites. By comparing mRNA-AGO2 interaction, miRNA
expression, and mRNA expression in MCF7 and MDA-MB231 cells, we identified a panel of mRNAs that were targeted by
miRNAs in a cell type-specific manner. We provided experimental evidence suggesting that cell type-specific usage of
alternative polyadenylation may be responsible for differential
SEPTEMBER 20, 2013 • VOLUME 288 • NUMBER 38
regulation by miRNAs of some mRNAs, such as RTPRK,
PMAIP1, and MAPK6.
In conclusion, we conducted a genome-wide analysis of
miRNA targets in luminal and basal-like breast cancer cells,
followed by experimental validation in cells with impaired
miRNA function at the level of single miRNA or global miRNA
processing. Our results suggest that miRNAs play an important
role in protecting cells against cell death and repressing metastasis.
We also provided experimental evidence supporting the possibility that alternative polyadenylation contributes to cell type-specific regulation of certain mRNAs by miRNA. These data provide
an overview of the function of endogenous miRNAs in two major
subtypes of breast cancer and a base of future studies to link breast
cancer cell properties with individual miRNAs.
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Comprehensive Analysis of MicroRNA
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Meiyun Fan, Raisa Krutilina, Jing Sun, Aarti
Sethuraman, Chuan He Yang, Zhao-hui Wu,
Junming Yue and Lawrence M. Pfeffer
J. Biol. Chem. 2013, 288:27480-27493.
doi: 10.1074/jbc.M113.491803 originally published online August 6, 2013
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