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Lentivirus-Mediated Small Interfering RNA Targeting VEGF-C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma.

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THE ANATOMICAL RECORD 292:633–639 (2009)
Lentivirus-Mediated Small Interfering
RNA Targeting VEGF-C Inhibited
Tumor Lymphangiogenesis and
Growth in Breast Carcinoma
Department of General Surgery, The Second Clinical Hospital,
Harbin Medical University, Harbin, China
Department of Anatomy, Harbin Medical University, Harbin, China
Lymph node metastasis is a major prognostic factor for patients with
breast cancer. The activation of vascular endothelial growth factor
(VEGF)-C plays a key role in lymph node metastasis through promoting
lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF-C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus-based VEGF-C siRNA
vector was infected into breast cancer cells and a xenograft model. The
expression of VEGF-C mRNA and protein were quantified by quantitative
real-time polymerase chain reaction (QRT-PCR), immunohistochemistry,
and western blot analysis. The effect of VEGF-C siRNA on breast cancer
cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti-LYVE-1 and anti-D2-40 by immunohistochemical analysis.
Lentivirus-mediated VEGF-C siRNA stably reduced VEGF-C mRNA and
protein expression. VEGF-C siRNA inhibited the invasive ability of breast
cancer cells in vitro. Five weeks after intratumoral injection, the tumor
volume was significantly smaller in the VEGF-C siRNA group than in the
control scramble siRNA group in the MDA-MB-231 cell xenograft model.
The numbers of LYVE-1 and D2-40 positive vessels per microscopic field
were significantly decreased in the VEGF-C siRNA group, which indicates
that VEGF-C siRNA inhibited lymphangiogenesis. Moreover, lymph node
metastasis was significantly suppressed by VEGF-C siRNA in vivo. In
conclusion, these results indicate that lentivirus-mediated VEGF-C
siRNA offers a new approach for therapeutic intervention to prevent
tumor growth and lymphatic metastasis of breast cancer. Anat Rec,
C 2009 Wiley-Liss, Inc.
292:633–639, 2009. V
Key words: VEGF-C; RNA interference; lymph node metastasis;
breast cancer
Drs. Baoliang Guo and Yafang Zhang contributed equally to
this work.
Grant sponsor: National Natural Science Foundation of
China; Grant number: 30672420; Grant sponsor: Department of
Education of Heilongjiang Province Foundation of China; Grant
number: No.1053G019.
*Correspondence to: Jianguo Zhang, M.D., Department of
General Surgery, The Second Clinical Hospital, Harbin Medical
University, 246 Xuefu Road, Harbin 150081, China.
Fax: þ86-451-86605079. E-mail:
Received 5 December 2008; Accepted 29 January 2009
DOI 10.1002/ar.20893
Published online in Wiley InterScience (www.interscience.wiley.
The spread of tumor cells to regional lymph nodes
through the lymphatic system plays a central role in the
dissemination of various human cancers such as breast
cancer (Achen et al., 2006). Experimental tumor models
and human clinicopathologic data indicate that lymphangiogenesis and lymph node metastasis are important prognostic indicators for the spread of cancer
(Eccles et al., 2007; Miyahara et al., 2007). Moreover,
lymphangiogenesis induced by breast cancer correlates
with lymph node metastasis and promotes metastasis
(Skobe et al., 2001).
Lymphangiogenic factors have been shown to stimulate tumor lymphangiogenesis and correlate with lymph
node metastasis (Stacker et al., 2002; Mohammed et al.,
2007). Among them, VEGF-C appears to be the most
important factor that contributes to lymphangiogenesis
and acts via the cognate receptor tyrosine kinase VEGF
receptor-3 (VEGFR-3) located on lymphatic endothelial
cells (Achen and Stacker, 2008). Increased VEGF-C
expression can promote the formation of tumor lymphangiogenesis and increase the metastatic spread of tumor
cells to lymph nodes in numerous cancers including
breast cancer (Mattila et al., 2002; Tamura and Ohta,
2003; Liu et al., 2008). Transgenic and knock-out mice
tumor models indicate that VEGF-C is a vital factor in
tumor lymphangiogenesis and the subsequent formation
of lymph node metastasis (Mandriota et al., 2001;
Karkkainen et al., 2004). Furthermore, overexpression
of VEGF-C in the tumor microenvironment has been
reported to be associated with poor prognosis and lymph
node metastasis in human breast cancer patients
(Nakamura et al., 2003; Mylona et al., 2007). In addition, tumor-secreted VEGF-C has recently been shown
to act systemically by inducing lymphangiogenesis in the
sentinel lymph node even prior to tumor cell invasion
(Hirakawa et al., 2007).
Small RNA interference (siRNA) technology has very
rapidly emerged as a revolutionary tool for elucidating
gene functions and offers a potential therapeutic strategy for various diseases including cancers (Dykxhoorn
and Lieberman, 2005; Izquierdo, 2005). In this study,
lentiviral-mediated siRNA targeting VEGF-C vector was
used to maintain high-transfection efficiency. We investigated the effect of VEGF-C siRNA on the invasive ability
of breast cancer cells in vitro and on lymphangiogenesis
and lymph node metastasis in vivo. Moreover, tumor
growth was examined in a xenograft model with subcutaneous implantation of MDA-MB-231 cells. Our results
indicate that lentivirus-mediated VEGF-C siRNA
decreased tumor invasion in vitro and inhibited lymphangiogenesis and lymph node metastasis in vivo. Lentivirus-mediated VEGF-C siRNA also inhibited growth
of the primary tumors in vivo. These results indicate
that gene therapy targeting VEGF-C by siRNA provides
a novel approach for the treatment of metastatic breast
100 U/mL penicillin. Cells were cultured in a humidified
37 C incubator with 5% CO2, fed every 2 days with complete medium containing 10% FBS, and then subcultured when confluence was reached.
Construction of lentiviral vectors and transfection. Lentivirus vectors for human VEGF-C small
hairpin RNA (shRNA) encoding a green fluorescent protein (GFP) sequence was constructed by GENECHEM
(Shanghai, China). The target shRNA sequence is 50 GGAGGCTGGCAACATAACA-30 (human VEGF-C gene
GenBank accession no. NM_005429), and four RNAi candidate target sequences to human VEGF-C were
designed. The lentivirus vectors containing VEGF-C
shRNA were constructed by ligating the Xho I/Sac II
digests of pEGFP-N1 and the VEGF-C shRNA PCR
product and were confirmed by sequencing. After testing
knockdown efficiencies in 293T cells by western blotting,
the lentivirus vectors with the best interference efficiency were selected to knockdown the endogenous
VEGF-C in breast cancer cells. Negative control shRNA
was provided by GENECHEM (Shanghai, China). Lentivirus-encoded shRNA against VEGF-C and control were
prepared and titered to 2 108 (TU/mL) as previously
described (Li and Rossi, 2008). Cells (1 104 cells/well)
were seeded in six-well plates overnight before transfection. The virus (0.1 mL) was mixed with 0.1 mL complete medium containing polybrene (8 mg/mL) and
added to cells and incubated for 1 hr at 37 C. Then, the
cells were incubated in fresh complete medium containing polybrene for 24 hr, followed by incubation for 48 hr
in complete RPMI-1640 medium. The cells were then
harvested for subsequent studies.
Quantitative real-time PCR. Total RNA was isolated using Trizol reagent (Promega) according to the
manufacturer’s instructions. One microgram of total
RNA was reverse transcribed into cDNA with Moloney
murine leukemia virus RT (Promega). Human GAPDH
RNA was used as an internal control. Primers for
VEGF-C and GAPDH are as follows: VEGF-C: forward:
CTTCCAGG-30 , reverse: 50 -ATGAGTCCTTCCACGATAC30 . Gene expression levels were evaluated by real-time
quantitative PCR kinetics with SYBR Master Mix
(Takara, Japan). Real-time PCR was performed with
1.0 lL of appropriate diluted cDNA, 0.5 lL (5 lM) of forward and reverse primers specific for human VEGF-C
and GAPDH, 10 lL of SYBR PremixEx taq, and 8.0 lL
of water. The cycling conditions were as follows: 95 C for
15 sec; 45 cycles of 95 C for 5 sec, and 60 C for 30 sec.
At the end of the cycles, the temperature was raised to
95 C for 1 min. The melting curve was achieved by first
cooling samples to 55 C for 1 min, and then followed by
81 cycles (30 sec/cycle), in which the temperature was
raised by 0.5 C per cycle to a maximum temperature
of 95 C.
Cell culture. The human breast cancer cell line
MDA-MB-231 was obtained from the American Type
Culture Collection. Cells were maintained in RPMI1640 (Solarbio, Beijing, China) supplemented with 10%
fetal bovine serum (FBS), 100 lg/mL streptomycin, and
Western blot analysis. Cells were lysed in ice-cold
lysis buffer containing 100 mM Tris-HCl (pH 6.8), 4%
sodium dodecyl sulfate (SDS), 20% glycerol, and 2%
hydroxyethanal. Equal amounts of cell lysate protein
(20 lg of protein per lane) were subjected to SDS-
In Vitro
polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinyl difluoride (PVDF) membranes. For
immunoblot analysis, the membranes were probed with
specific primary antibodies: anti- VEGF-C, anti-Bcl-2,
anti-Bax, and anti-GAPDH antibody (Santa Cruz). The
bound primary antibodies were then incubated with
horseradish peroxidase-conjugated anti-goat or antimouse (Santa Cruz) secondary antibodies, and then developed with an enhanced chemiluminescence detection system according to the manufacturer’s instructions.
Invasion assay. The in vitro cell invasive assay was
done in modified Boyden chambers. The top and bottom
of the cell invasion chamber were separated by a polycarbonate filter with pores of 8-lm, which was coated
with Matrigel. Approximately 1 104 cells were suspended in 100 lL serum-free supplement and were seeded
onto the top chamber, and 1 mL of culture medium with
10% FBS was added to the lower chamber. Forty-eight
hours later, the cells that had migrated to the lower chamber were fixed in methanol and stained with hematoxylin.
The invasive activity of cancer cells was determined by
counting the cells with a microscope at 200 magnification. Five random visual fields were counted for each well,
and the average was determined.
In Vivo
Animals and tumor production. Four- to 5-weekold female nu/nu nude mice were purchased from Beijing
Vital River Experimental Animals Co. (Beijing, China)
and housed in the Experimental Center of the Harbin
Medical University. All animal protocols used for this
study were approved by the Institutional Animal Care
and Use Committee. To produce tumors, MDA-MB-231
cells (1 106 cells) in 0.1 mL HBSS were implanted into
the left inguinal mammary fat pads of mice. Mice were
randomized into three groups (six mice/group). After
1 week, one group of nude mice was intratumorally
injected with PBS, the second group with lentivirus
scramble siRNA, and the third group with lentivirus
VEGF-C siRNA. Mice in all groups were injected weekly
until the completion of experiments. Tumor growth was
monitored weekly and measured in two dimensions.
Tumor volume was calculated using the formula V ¼
W2 L/2, where W and L are the shortest and longest
diameters, respectively. After 5 weeks, mice were killed
and tumors were fixed in 10% formalin solution and
processed for immunohistochemical analysis of lymphangiogenesis and lymph node metastasis.
Immunohistochemical analysis. Immunohistochemical staining was performed using the streptavidinperoxidase conjugate method. Briefly, serial 4-lm-thick
sections were cut from formalin-fixed and paraffinembedded tumor blocks, dewaxed in xylene, rehydrated
through sequential changes of alcohol, and then incubated with fresh 3% hydrogen peroxide for 15 min at
room temperature. After washing with phosphate-buffered saline (PBS), the tissue sections were antigenretrieved in 0.01 M citrate buffer, pH 6.0, in a pressure
steamer for 10 min. Sections were blocked with appropriate normal serum in PBS. After washing with PBS,
slides were incubated overnight at 4 C with VEGF-C
polyclonal rabbit anti-human antibody (Zhongshan Bio-
technology, Beijing, China), LYVE-1 monoclonal antimouse LYVE-1 antibody (R&D Systems, Minneapolis,
MN) or D2-40 monoclonal anti-mouse antibody (Santa
Cruz Biotechnology). After washing with PBS, slides
were incubated with polyHRP goat anti-rabbit IgG
(Zhongshan Biotechnology, Beijing, China) for 25 min at
room temperature. After washing with PBS, slides were
stained with fresh 3,30 -diaminobenzidine (DAB; Zhongshan Biotechnology, Beijing, China), counterstained in
hematoxylin, dehydrated, and mounted. Sections of axillary lymph nodes were stained with anti-pan-cytokeratin
antibody (Santa Cruz Biotechnology). The number of
positive vessels was counted at 400 magnification in
the area with highest intensity. Six different fields were
quantified for each sample and the average value was
calculated. Each section was examined by two independent observers.
Statistical analysis. Data are expressed as means SE. The significance of the data was determined by
Student’s t test (two-tailed) and ANOVA analysis for all
in vitro studies and the Mann-Whitney U test for in vivo
studies. A P-value < 0.05 was considered statistically significant. All statistical analyses were done using SPSS
Effect of VEGF-C siRNA on VEGF-C Expression
Multiple siRNA sequences were designed, and the lentivirus siRNA vector with the greatest inhibitory activity
was chosen. As shown in Fig. 1A, the real-time RT-PCR
results indicate that endogenous VEGF-C mRNA expression was significantly inhibited at 24 hr after infection
in MDA-MB-231 cells. Compared with the control siRNA
group, the VEGF-C siRNA group showed relatively
lower quantities of VEGF-C mRNA; mRNA expression
was decreased by nearly 70% (Fig. 1A). In accordance
with this, Western blotting showed that VEGF-C protein
expression was suppressed in the VEGF-C siRNA group
(by 48%) compared with the control siRNA group in
MDA-MB-231 cells (Fig. 1B). Infection success was also
demonstrated by visual inspection for green fluorescence. The transfection rate was calculated by the percentage of fluorescent cells in the total cells and was
over 70% in each visual field (Fig. 1C).
Effect of VEGF-C siRNA on Cell Invasion and
Bcl-2/Bax Protein Expression
To determine the function of lentivirus-mediated
VEGF-C siRNA, MDA-MB-231 cells, a highly metastatic
breast cancer cell line, were used. Using a Boyden chamber, we determined changes in cell invasion after 48 hr.
The cells were fixed and stained with crystal violet to
determine the number of cells that invaded across the
membrane. Compared with the control siRNA infected
cells, VEGF-C siRNA infected cells showed a significant
decrease in invasion (Fig. 2A). Colorimetric analysis of
the crystal violet-stained migratory cells indicated a 52%
decrease in the number of invasive VEGF-C siRNA
infected cells compared with control siRNA infected
cells. Next, we also observed the effect of VEGF-C siRNA
on Bcl-2 and Bax protein expression. Results showed
Fig. 1. Downregulating VEGF-C expression by lentivirus-mediated
VEGF-C siRNA in MDA-MB-231 cells. MDA-MB-231 cells were
infected with lentivirus-based VEGF-C siRNA and control scrambled
siRNA. VEGF-C mRNA (A) and protein (B) expression were significantly suppressed in the VEGF-C siRNA group compared with the
control scramble siRNA group. Representative images were taken by
fluorescence microscope at 24 hr after infection (C). The green fluorescence (GFP) demonstrates successful infection. A representative
image for one of three independent experiments is shown. * Significantly different from scramble siRNA (P < 0.05).
that VEGF-C siRNA decreased the ratio of Bcl2/Bax
(Fig. 2B).
was observed in the mouse experiments, as assessed by
changes in behavior, appearance, or weight.
Effect of VEGF-C siRNA on Primary
Tumor Growth
Effect of VEGF-C siRNA on Tumor
We next examined the effect of lentivirus-mediated
VEGF-C siRNA on murine mammary tumor growth. As
shown in Fig. 3, at day 35, the xenograft tumor sizes are
as follows (mean SE): 915 186 mm3 in the PBS
group, 869 165 mm3 in the scramble siRNA group,
and 422 104 mm3 in the VEGF-C siRNA group.
ANOVA analysis revealed that the inhibitory effect
exerted by siRNA infection in the xenografts was significant compared with that for scramble siRNA or for PBS
(P < 0.05). Compared with the starting volume, delayed
tumor growth was significant in the VEGF-C siRNA
group and was evident from the day after starting therapy until the day the mice were sacrificed. No toxicity
Positive immunostaining for VEGF-C was observed in
the cytoplasm of the tumor cells and the expression level
was significantly decreased in the VEGF-C siRNA group
compared with the scramble siRNA and PBS groups (Fig.
4A). Next, the effect of VEGF-C siRNA on lymphangiogenesis was determined by immunohistochemical analysis
using anti-LYVE-1 and anti-D2-40 antibodies. Representative results and quantitative data from the microlymphatic density analysis are shown in Fig. 4B,C,
respectively. The numbers of LYVE-1 and D2-40 positive
cells per microscopic field were 3.78 1.1 and 3.76 1.0
in VEGF-C siRNA group, compared with 7.48 1.0 and
7.48 0.9 per microscopic field in the scramble siRNA
Fig. 2. Inhibiton of breast cancer cell invasive ability by lentivirusmediated VEGF-C siRNA. A cell invasion assay of breast cancer cells
was performed using a cell invasion assay kit. MDA-MB-231 cells
were infected with VEGF-C siRNA or control scramble siRNA. The rate
of cell invasion was evaluated in a modified Boyden chamber assay
as described in the Materials and Methods. Histograms show the percentage of invading cells, with the invasion of cells in the scramble
siRNA group designated as 100%. Data are representative of three
independent experiments (A). The ratio of Bcl-2 and Bax protein (B)
expression were significantly suppressed in the VEGF-C siRNA group
compared with the scramble siRNA group. * Significantly different
from scramble siRNA (P < 0.05).
group and 7.55 0.9 and 7.62 0.9 per microscopic field
in the PBS group, respectively (Fig. 4). These data suggest
that inhibition of VEGF-C in primary tumors by VEGF-C
siRNA decreases lymphangiogenesis.
Effect of VEGF-C siRNA on Local Lymph
Node Metastasis
Finally, we investigated the effect of VEGF-C siRNA
on local lymph node metastasis. We found that nearly
all of the control mice treated without VEGF-C siRNA
developed metastasis in the lymph nodes, whereas some
mice in VEGF-C siRNA group did not have lymph node
metastases. The incidence of lymph node metastasis was
significantly decreased to 42% in the VEGF-C siRNA
group compared with 81% in the scramble siRNA group
and 83% in PBS group. These data suggest that inhibition of VEGF-C in primary tumors by VEGF-C siRNA
suppressed local lymph node metastasis. In addition, the
frequency of pan-cytokeratin-positive metastatic cells in
lymph nodes was significantly lower in the VEGF-CsiRNA group compared with the control groups (Fig. 5).
In this study, VEGF-C mRNA and protein expression
was suppressed by lentivirus-mediated VEGF-C siRNA.
VEGF-C siRNA inhibited the tumor invasion ability and
the ratio of Bcl-2/Bax in vitro. Lentivirus-mediated
VEGF-C siRNA inhibited lymphangiogenesis and spontaneous lymph node metastasis in the MDA-MB-231 cell
xenograft model. Furthermore, lentivirus-mediated
VEGF-C siRNA inhibited primary tumor growth of breast
cancer in vivo.
Breast cancer is the most common malignant tumor
and metastasis of tumor cells is the second leading cause
of cancer death affecting women worldwide (Stewart and
Kleihues, 2003). Although tumor cell dissemination is
mediated by a number of mechanisms, the most common
Fig. 3. Suppression of tumor growth by lentivirus-mediated VEGFC siRNA in MDA-MB-231 cell xenografts. MDA-MB-231 cell xenograft
mice were intratumorally injected with lentivirus-mediated VEGF-C
siRNA, scramble siRNA, or PBS. The size of the primary tumors was
measured every week. Mice were sacrificed after 5 weeks. Points,
mean tumor volume in each group; bars, SE. * Significantly different
from scramble siRNA (P < 0.05).
pathway of initial dissemination is through the lymphatic system to the regional lymph nodes (Pepper
et al., 2000). The presence of metastases in regional
lymph nodes is a strong indicator of low-patient survival
rate, poor patient prognosis in many types of cancer
(Skobe et al., 2001; Miyahara et al., 2007; Liu et al.,
2008). Despite advances in the treatment of breast cancer, morbidity and mortality from breast cancer remain
unacceptably high. Nevertheless, more novel targets for
therapeutic development have been identified and are
being explored.
Recent studies have demonstrated that VEGF-C overexpression is associated with lymph node metastasis by
enhancing intratumoral lymphangiogenesis in breast
carcinoma (Skobe et al., 2001; Mattila et al., 2002).
Experiments with transgenic mice have shown that
overexpression of VEGF-C results in lymphatic endothelial proliferation and vessel enlargement (Jeltsch et al.,
1997). Likewise, the clinical significance of VEGF-C
expression in relation to lymph node metastasis and
patient outcome has been reported in cancers of the
esophagus, stomach, and colorectum (Liu et al., 2008).
Downregulation of VEGF-C or blockade of VEGFR-3
signaling inhibits lymphangiogenesis and lymph node
metastasis in experimental animal cancer models (Chen
et al., 2005; Lin et al., 2005; Burton et al., 2008). These
results indicate that VEGF-C could be an important
therapeutic target to suppress lymphangiogenesis and
lymphatic metastasis. Consists with previous study, our
results showed VEGF-C siRNA might be exerted its
function by decreasing the ratio of Bcl-2/Bax protein
SiRNA technology provides a novel strategy for investigating gene expression and function (Bantounas et al.,
2004). Compared with antisense oligonucleotides and
ribozymes, siRNA allows us to target any gene with
greater specificity and efficiency, and it can be developed
in various ways allowing for numerous in vitro and
in vivo applications (Elbashir et al., 2001; Chen et al.,
Fig. 4. Suppression of tumor lymphangiogenesis by lentivirus-mediated VEGF-C siRNA in MDA-MB-231 cell xenografts. Tumors were
harvested and immunostained with VEGF-C, LYVE-1 and D2-40 antibody to assess lymphangiogenesis, as described in the Materials and
Methods. VEGF-C expression in tumor cells (A), LYVE-1-positive
microlymphatic vessel (B), and D2-40-positive microlymphatics (C)
were decreased in the VEGF-C-siRNA group compared with the control and scramble siRNA groups (original magnification 400). Scale
bar of immunostain results was shown in Fig.4D. * Significantly different from scramble siRNA.
2003; Song et al., 2003). In addition, siRNA is becoming
an important tool for the study of biological processes
and has the potential for therapeutic applications in
human cancer diseases (Dykxhoorn et al., 2006; Takeshita and Ochiya, 2006). Thus, we used siRNA to assess
the effect of suppressing the VEGF-C gene in lymphatic
metastasis in breast cancer.
Effective delivery of siRNA to target cells is still a
major issue. The use of siRNA/shRNA for gene knockout
requires an efficient stable transfection or transduction
process. To improve efficiency, a lentivirus vector-based
siRNA was constructed instead of synthetic RNA oligonucleotides or plasmid-encoding shRNAs (An et al.,
2003). In this study, lentivirus vector delivery produced
high-transfection rate in vitro and in vivo. Compared
with the other vectors, the lentivirus-based gene vectors
had advantages over the other viral and nonviral vectors
in that they can be delivered to and integrate with
genetic material in cells that are dividing or not (Cockrell and Kafri, 2007). Thus lentivirus-mediated siRNA
targeting of VEGF-C appears to be particularly efficacious to ensure gene silencing in vitro and in vivo.
Fig. 5. Suppression of local lymph node metastasis by lentivirusmediated VEGF-C siRNA in MDA-MB-231 cell xenografts. Mice were
sacrificed after 5 weeks and spontaneous metastatic lymph nodes
were fixed. Lymph node metastases were examined by immunohistochemistry with pan-cytokeratin staining. Columns represent the incidence and frequency of pan-cytokeratin-positive cells in the lymph
nodes. * Significantly different from scramble siRNA (P < 0.05).
Taken together, our results demonstrate that lentivirus-mediated VEGF-C siRNA inhibited lymphangiogenesis, lymph node metastasis and primary tumor growth
in MDA-MB-231 cell xenografts. These results indicate
that lentivirus-mediated siRNA targeting VEGF-C offers
a therapeutic strategy for inhibiting lymphatic metastasis and for the treatment of breast carcinoma.
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