Introduction
Breast cancer is the most common type of cancer in
women. It is known that cancer stem cells have critical roles in
breast cancer progression. CD44 is considered to be a stem cell
marker for breast cancer (1) and
has important roles in processes of cancer progression, including
proliferation, cell cycle, stem cell properties and metastasis
(2,3). The cancer stem cell (CSC)
sub-population is, at least in part, responsible for therapy
resistance and metastasis of cancer cells (4). Studies have shown that CSCs and cells
undergoing epithelial-mesenchymal transition (EMT), which have
CSC-like properties, have crucial roles in drug resistance and
early cancer metastasis of numerous cancer types, including breast
cancer (5–8). In spite of the abundance of studies
on CSCs, the molecular mechanisms associated with their role in
tumor progression and resistance require further study.
Emerging evidence indicated the critical roles of
microRNAs (miRNAs/miRs) in cancer (9). miRNAs are small and non-coding
nucleotides which regulate gene expression by imperfectly binding
to the 3′-untranslated regions (3′UTR) of target mRNAs, leading to
the inhibition of their translation (10). miRNAs are known as important
factors regulating organ development, cell proliferation,
differentiation, apoptosis and cell cycle (9,10).
In the recent years, an increasing number of miRNAs have been
identified to be aberrantly up- or downregulated in various types
of cancer, and to be either positively or negatively associated
with their development and progression (9,10).
A previous microarray study performed by our group
showed that several stem cell markers, such as CD44, were
upregulated in transplanted breast cancers (unpublished data). As
CD44 was increased to a greater extent than the other stem cell
markers, the present study isolated CD44+ and CD44+ cells to assess
differences in their tumorigenicity. A miRNA array performed in
CD44+ breast cancer cells demonstrated that miR-143 was markedly
downregulated. Therefore, the present study assessed the effects of
miR-143 targeting CD44 on cellular properties associated with
metastasis and cancer progression. The present study suggested that
miR-143 functions as a tumor suppressor in breast cancer.
Materials and methods
Cell culture
The breast cancer cell lines BT474, MDA231, MDA468,
MCF7, SKBR3, and SUM102 were obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA). The cells were
cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL;
Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine
serum, penicillin (100 U/ml) and streptomycin (100 μg/ml)
(all from Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured
in a humidified atmosphere containing 5% CO2 at 37°C.
The MCF10A normal human breast cell line was purchased from ATCC.
MCF10A cells were cultured in DMEM/F-12 containing 5% horse serum
(Thermo Fisher Scientific), 100 U/ml penicillin, 100 μg/ml
streptomycin, 10 μg/ml insulin (Sigma-Aldrich) and 20 ng/ml
epidermal growth factor (R&D Systems, Minneapolis, MN,
USA).
Clinical specimens
Primary breast cancer tissue samples and adjacent
normal tissue samples were obtained at the Third Affiliated
Hospital of Kunming Medical University (Kunming, Yunnan) from 2006
to 2011. None of the patients (mean age, 51.3 years) received
chemotherapy or radiotherapy prior to surgery. Hematoxylin and
eosin staining was used to confirm the diagnosis of breast cancer
(11–13). Tissue collection protocols were
approved by the Ethics Committee of the First Affiliated Hospital
of Soochow University (Suzhou, China).
miRNA precursor expression profiling
Total RNA was isolated from the CD44+ and CD44−
breast cancer cells using TRIzol (Invitrogen; Thermo Fisher
Scientific). The RNA concentration was quantified using the ND-1000
micro-spectrophotometer (NanoDrop; Thermo Fisher Scientific). The
integrity of the RNA was evaluated using the Agilent 2100
Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Total RNA
was used for reverse-transcription (RT) reaction to produce cDNA
using gene-specific primers to 222 miRNA precursors plus 18S
ribonucleic (r)RNA. Real-time quantitative polymerase chain
reaction (qPCR) was used to examine the expression of the 222 miRNA
precursors. All primers and all components of the PCR mixtures were
provided by Shanghai Kangcheng (Shanghai, China) and reactions were
performed in an ABI Prism® 7900HT real-time PCR
apparatus (Thermo Fisher Scientific). The thermocycling conditions
were as follows: 1 cycle at 50°C for 2 min, 1 cycle at 95°C 10 min
and 95°C for 15 sec and 60°C for 30 sec (40 cycles). The mean cycle
threshold (CT) value was determined from duplicate PCRs. The amount
of miRNAs, was normalized to 18S rRNA and the relative gene
expression was calculated as 2−[CT(miRNA) − CT(18S
rRNA)].
Plasmid construction
To construct the miR-143 expression vector
(P-miR-143), a fragment including the mature miR-143 sequence was
amplified from MRC-5-cell genomic DNA (Biomat, Bejing, China) and
then cloned into the BamHI and XbaI sites in pcDNA3.1
(Invitrogen). The following primers were used: Forward,
5′-CGGCTAGCCGTTCAGTTTCTAAACCAGCAC-3′ and reverse,
5′-GCTCTAGAGCCTACATGGCATCATCCTTC-3′ (Invitrogen). CD44 was
predicted as the target gene of miR-143 using TargetScan
(http://www.targetscan.org/vert_61/).
The 3′UTR of CD44 was amplified from MRC-5 cells by PCR (11). The primers for CD44 were as
follows: Forward, 5′-GTTCCTGGACTGATTTC-3′ and reverse,
5′-TTACTCTGCTGCGTTG-3′ (Invitrogen). The thermocycling conditions
were 94°C for 5 min followed by 25 cycles of 94°C for 30 sec, 55°C
for 30 sec and 72°C for 1 min, and a final extension at 72°C, for
10 min. These fragments were ligated into the NheI and
XbaI sites with specific restriction enzymes (Promega Corp.,
Madison, WI, USA) based on the pmirGLO Dual-Luciferase miRNA Target
Expression Vector (Promega Corp.), which included firefly
luciferase and renilla luciferase. Firefly luciferase is a reporter
monitoring mRNA regulation and renilla luciferase is a control
reporter for normalization. The mutant 3′-UTR of CD44 was generated
using the overlap-extension PCR method (RiboBio Co. Ltd.,
Guangzhou, China). All constructs were confirmed by DNA sequencing
analysis (ABI3730 DNA sequencer; Thermo Fisher Scientific). For
each group, 2×105 SKBR3 cells were transfected with
miR-143 or CD44 using Lipofectamine 2000 (Invitrogen) according to
the manufacturer's instructions. The miR-143 mimic, a nonspecific
miR control, anti-miR143a and a nonspecific anti-miR control were
all purchased from Sigma-Aldrich.
RNA isolation and RT-qPCR
The tissues were homogenized using TissueLyser II
(Qiagen, Hilden, Germany) with natural extraction buffer and 10%
sodium dodecyl sulfate (SDS). Total RNA from the tissues or cells
transfected with miRNA-expressing plasmids was extracted using
TRIzol (Invitrogen) according to the manufacturer's instructions.
RT-qPCR was performed using Real-Time RT-PCR Master Mix containing
SYBR Green I and hotstart Taq DNA polymerase (Toyobo, Osaka,
Japan). CD44, CD133, c-myc and GAPDH primers were supplied by
Sangon Biotech, Co., Ltd. (Shanghai, China) with the following
sequences: miR-143 forward, 5′-TGAGATGAAGCACTGTAGCTC-3′ and
reverse, 5′-GCGAGCACAGAATTAATACGAC-3′; CD44 forward,
5′-GGACTCTGCCTCGTGCCG-3′ and reverse, 5′-ATTGAAGCAATATGTGTCATAC-3′;
CD133 forward, 5′-ACTCGGCTCCCTGTTGCTGCT-3′ and reverse,
5′-GAGCCTGATATCCTGACCATTG-3′; OCT4 forward,
5′-GTCTTCTGCCTTTTAAAATCCA-3′ and reverse,
5′-ACTCGGTTCTCGATACTGGTT-3′; GAPDH forward,
5′-GGAAGGTGAAGGTCGGAGTCA-3′ and reverse,
5′-GGAAGATGGTGATGGGATTTC-3′; and U6 small nuclear (sn)RNA forward,
5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCCT-3′.
PCR was performed in an ABI Prism® 7900HT real-time PCR
apparatus with the following conditions: 95°C for 1 min; followed
by 40 cycles of 95°C for 10 sec and 65°C for 40 sec. Relative gene
expression was determined using the ΔΔCT method with normalization
to the housekeeping gene GAPDH or U6 snRNA using the formula
2−ΔΔCT.
Fluorescence-activated cell sorting
(FACS)
Cells (104) were suspended in FACS buffer
[phosphate-buffered saline (PBS) containing 0.1% bovine serum
albumin and 0.1% Triton X100] and incubated in fluorescein
isothiocyanate-conjugated anti-CD44 antibody for 15 min at room
temperature. Cells were then washed with PBS and re-suspended in
PBS for FACS analysis (6HT; Guava®; Merck-Millipore,
Billerica, MA, USA) and to isolate CD44+ and CD44− negative
cells.
MTT assay
Breast cancer cells were transfected with
miRNA-containing plasmids for 24 h, collected and re-seeded in a
96-well plate at 5×103 cells per well. The control group
was transfected with control miRNA. The breast cancer cells were
incubated at 37°C for 0, 1, 2, 3, 4 or 5 days and 10 μl MTT
(5 mg/ml; Sigma-Aldrich) was added, followed by further culture for
4 h. The medium was discarded and 150 μl dimethyl sulfoxide
(Sangon, Shanghai, China) was added to each well to dissolve the
formazan crystals completely. The absorbance at 570 nm was measured
using an ELISA reader (SM800; Utrao Medical Instruments, Shanghai,
China).
Colony formation assay
Breast cancer cells were transfected with
miRNA-containing plasmids for 24 h in six-well plates, digested
with 0.1% trypsin containing 0.02% EDTA and re-suspended in medium
at 105 cells/ml. A total of 100 cells were added to each
well of six-well plates and incubated at 37°C for 24 h. At day 3,
the medium was replaced. After 10 days, colonies which had formed
(comprising at least 50 cells) were counted under a microscope
(IX73; Olympus, Tokyo, Japan).
Tumor spheroid assay
Breast cancer cells were transfected with miRNAs or
plasmids for 24 h in six-well plates. Single-cell suspensions were
prepared and cells were plated on six-well ultralow attachment
plates (Corning-Costar Inc., Corning, NY, USA) at a density of
1,000 cells/ml. Cells were grown in DMEM supplemented with 1% N2
Supplement (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/ml
human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal
growth factor (Invitrogen) and 1% antibiotic-antimycotic
(Invitrogen). After 10 days of culturing, the mammospheres were
counted.
Western blot analysis
Total protein from the cells transfected with miRNAs
or plasmids was extracted by lysis in radioimmunoprecipitation
assay buffer containing 1X protease inhibitor cocktail (both from
Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and quantified
using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA,
USA). Equal amounts of protein (50 μg/lane) were separated
by 12.5% SDS-PAGE and then transferred onto polyvinylidene
difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 55 V
for 4 h at 4°C. The protein in the PVDF membranes was blocked in 5%
fat-free dry milk in Tris-buffered saline (TBS). The PVDF membranes
were incubated with primary antibodies in TBS containing Tween 20
(TBST; Sangon) overnight at 4°C and washed three times with TBST,
followed by incubation with horseradish peroxidase (HRP)-conjugated
secondary antibodies in TBS for 1 h at room temperature. The
following antibodies were used: CD44 (156-3C11) mouse monoclonal
antibody (cat. no. 3570; dilution, 1:1,000; Cell Signaling
Technology, Danvers, MA, USA), fibronectin antibody (A-11; (cat.
no. sc-271098; dilution, 1:500), E-cadherin antibody (67A4; cat.
no. sc-21791; dilution, 1:1,000) and vimentin antibody (J144; cat.
no. sc-53464; dilution, 1:1,000) (all from Santa Cruz
Biotechnology, Inc) as well as anti-αSMA (1A4; dilution, 1:2,000;
Sigma-Aldrich). As secondary antibodies, HRP-conjugated goat
anti-mouse immunoglobulin G (IgG) (cat no. sc-2005; dilution,
1:3,000) and HRP-conjugated goat anti-rabbit IgG (cat no. sc-2004;
dilution, 1:2,000) were used (both from Santa Cruz Biotechnology,
Inc.). Protein bands were visualized using an enhanced
chemiluminescence Western Blotting kit (Pierce Biotechnology, Inc.,
Rockford, IL, USA). Protein bands were captured using a Canon
CanoScan LiDE 220 image scanner (Canon, Tokyo, Japan).
Transwell invasion assay
Breast cancer cells were transfected with
miRNA-containing plasmids in six-well plates for 24 h and then
collected for a migration assay. A total of 1×105 breast
cancer cells were seeded in the upper chambers of a Transwell
system with Matrigel-coated Membranes (24-well insert; pore size, 8
μm; Corning-Costar). The cells invaded through the
Matrigel-coated membrane were fixed with methanol, stained with
0.1% crystal violet (Sigma-Aldrich) and counted under a light
microscope (magnification, 40×; three random fields per well).
Mouse model of breast cancer
Six-week old female BALB/c nude mice (n=15) were
purchased from the Shanghai Laboratory Animal Center (Shanghai,
China). The animals were maintained under regulated pathogen-free
conditions, and kept with a 12-h light/dark cycle at 25°C and
40–60% relative humidity. Mice were treated with miRNAs or the
control via injection into the right flank. Tumor size was
calculated using a vernier caliper. The experiments were approved
by the Ethics Committee of Yunnan Tumor Hospital (Kunming, China).
To establish the mouse model of breast cancer, 2×105
SKBR3 cells transfected with miR-143-overexpressing vector were
inoculated subcutaneously into the dorsal flanks of the mice (five
per group). The tumor size was measured every seven days. At 28
days following inoculation the mice were sacrificed, and tumors
were harvested, weighed and measured. The tumor volumes were
calculated using the following formula: Largest diameter ×
(diameter perpendicular to the largest diameter)2/2. All
procedures involved were performed following institutional
guidelines.
Statistical analysis
Values are expressed as the mean ± standard
deviation. Associations between protein expression levels in the
breast tissue microarray were assessed using Spearman's rank
correlation test. Comparisons between different groups were
performed by one- or two-way analysis of variance, followed by
Bonferoni's multiple comparisons tests using GraphPad Prism
statistical analysis software (GraphPad Software, Inc., La Jolla,
CA, USA). P<0.05 was considered to indicate a statistically
significant difference between values.
Results
CD44 promotes progression of breast
cancer
Microarray data from a previous study performed by
our group (unpublished data) showed that expression levels of CD44,
CD133 and OCT4 were significantly increased in transplanted cells
compared to those in primary breast cancer tissues. In the present
study, overexpression of CD44 was verified using real-time RT-qPCR
(Fig 1A). CD44+ cells were
quantified and isolated by flow cytometry (Fig. 1B), revealing that CD44+ cells were
present at a larger proportion in transplanted tumor cells than in
primary tumor cells. Furthermore, the biological properties of
CD44+ cells were analyzed. The proliferation of CD44+ breast cancer
cells was increased compared with that of CD44− cells (Fig. 1C and D). In addition, the sphere
formation of CD44+ cells was increased compared with that of CD44−
cells (Fig. 1E). These results
indicated that CD44+ breast cancer cells had stem-like
properties.
miR-143 is downregulated in CD44+ breast
cancer cells
miRNA profiling was performed in CD44+ and CD44−
breast cancer cells, revealing that miR-143 was significantly
downregulated in CD44+ breast cancer cells compared to that in
CD44− cells (Fig. 2A). Next, the
expression of miR-143 in 16 human breast cancer samples and 16
normal tissues was examined by real time RT-qPCR. As shown in
Fig. 2B, miR-143 expression levels
in tumor samples were lower than those in normal tissues. In
analogy with this result, miR-143 expression was lower in human
breast cancer cell lines compared with that in a normal breast cell
line (Fig. 2C). These results
indicated that miR-143 may suppress breast cancer progression.
CD44 is a novel target gene of miR-143 in
breast cancer cells
To investigate the association of CD44 and miR-143,
a Targetscan analysis (http://www.targetscan.org/) was performed, which
indicated that CD44 may be directly suppressed by miR-143 (Fig. 3A). To experimentally verify this
interaction, a luciferase reporter assay was performed (Fig. 3B), which demonstrated that
following transfection with miR-143, the luciferase activity of a
reporter plasmid containing the wild-type 3′UTR of CD44 in CD44+
cells was lower than that in control cells, while luciferase
activity in cells transfected with a reporter plasmid containing a
mutated 3′UTR of CD44 was markedly elevated and not affected by
miR-143. Furthermore, RT-qPCR analysis demonstrated that compared
with the control, endogenous CD44 mRNA levels were downregulated in
the cells with miR-143 overexpression (Fig. 3C). In addition, western blot
analysis showed that following transfection of the CD44+ SKBR3
breast cancer cells with miR-143, CD44 was decreased (Fig 3D), revealing that miR-143 regulated
endogenous CD44 expression in breast cancer cells. These results
clearly indicated that CD44 is a novel target gene of miR-143.
miR-143 inhibits progression and stem
cell-like properties of breast cancer cells by targeting CD44
To explore the roles of miR-143 in cell survival and
proliferation, SKBR3 cells were infected with miR-143 plasmid or
control plasmid. A colony formation assay demonstrated that miR-143
inhibited the colony formation rate of SKBR3 cells (Fig. 4A). Furthermore, cell cycle analysis
indicated that SKBR3 cells with miR-143 expression showed a
decrease in the G1-phase population and an increase in S-phase
population (Fig. 4B). Next, the
self-renewal potential of breast cancer cells was examined. The
stem-cell sphere formation assay revealed that miR-143
overexpressing CD44+ cells showed a significantly decreased ability
of self renewal, with the sphere-formation rate being reduced by
87% (Fig. 4C). Furthermore, the
effects of miR-143 on breast cancer cells were assessed in
vivo using a mouse model. The tumor growth curve indicated that
miR-143 reduced breast cancer formation (Fig. 4D).
miR-143 inhibits breast cancer cell
metastasis by targeting CD44
To further elucidate the role of miR-143 in breast
cancer metastasis and EMT, CD44+ cells were infected with
miR-143-containing vector and subjected to a Transwell assay, which
indicated that upregulation of miR-143 significantly decreased the
invasive capacity of breast cancer cells (Fig. 5A and B). In addition, the
expression of EMT markers was assessed by western blot analysis.
The results showed that mesenchymal markers fibronectin, vimentin
and α-SMA were decreased, while epithelial marker E-cadherin was
increased in breast cancer cells with miR-143 overexpression
(Fig. 5C). These results indicated
that miR-143 suppressed metastasis and EMT of breast cancer
cells.
Discussion
Cell surface markers of CSCs can help distinguish,
isolate and purify these tumor-initiating cells for further
biological investigation. A previous study by our group showed a
upregulation of stem-cell markers in CSCs, including CD44
(unpublished data). The present study investigated cell surface
protein CD44, which is commonly used as a stem cell marker to
isolate breast cancer stem cells (1). A mammosphere formation assay was used
to show that the self-renewal ability of CD44+ breast cancer cells
in serum-free medium was higher than that in CD44− cells.
Furthermore, an MTT and a clonogenic assay revealed that CD44+
breast cancer cells possessed a greater viability and a higher
colony formation rate than CD44− cells. These experiments
demonstrated that CD44+ breast cancer cells exhibited higher
tumorigenicity and self-renewal ability than those of CD44− cells,
and that they had cancer stem-like properties.
Bioinformatics analysis revealed that CD44 was a
potential target gene of miR-143, which was experimentally verified
using a luciferase activity assay. Furthermore, it was demonstrated
that miR-143 inhibited breast cancer-cell proliferation as well as
their stem cell-like properties and EMT by down-regulating CD44.
The EMT is a key developmental process, which is frequently
activated during cancer-cell invasion and metastasis, and which may
promote resistance to chemotherapy (5). Loss of epithelial cell markers such
as E-cadherin is considered to be a major hallmark of the EMT
(5–8). E-cadherin is a classical member of
the cadherin superfamily, which maintains epithelial cell
plasticity. Mesenchymal cell markers usually induce EMT and
metastasis of cancer. Accumulating evidence suggested that the EMT
is closely associated with the acquisition of stem cell-like
properties of cancer cells (6).
The present study was the first to indicate that miR-143 suppresses
EMT of CD44+ breast cancer cells and breast cancer
tumorigenesis.
Previous studies indicated that low expression of
miR-143 is closely linked to cancer development and identified it
as a tumor suppressor (11–14).
miR-143 regulates various target genes, including Limk1 (15), SDC1 (16), COX-2 (17), PKCε (18), MACC1 (19) and KRAS (20), in non-small cell lung cancer,
melanoma, gastric cancer, breast cancer and prostate cancer. The
present study showed that miR-143 inhibited breast cancer via CD44
expression.
Previous studies have indicated that miR-143 could
suppress cell proliferation, glycolysis, metastasis and induce
apoptosis in cancer cells (15–20).
The present study demonstrated that miR-143 inhibited breast cancer
cell proliferation assayed by MTT, colony formation and mammosphere
formation by targeting CD44. The in vivo study also
demonstrated that miR-143 suppressed breast cancer growth. The
results also demonstrated that miR-143 inhibited breast cancer cell
metastasis, including invasion and EMT.
In conclusion, the present study indicated that
miR-143 functions as a tumor suppressor in breast cancer and that
low miR-143 expression in breast cancer tissues may be an
unfavorable prognostic factor. The association between miR-143 and
CD44 in breast cancer tissues requires further study. To the best
of our knowledge, the present study was the first to demonstrate
that downregulation of miR-143 increased CD44 expression in breast
cancer cells, which enhances their CSC-like properties; these are
linked with breast cancer progression, metastasis and resistance to
chemotherapy.
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