CD147 silencing via RNA interference reduces tumor cell invasion, metastasis and increases chemosensitivity in pancreatic cancer cells
CD147, which belongs to the immunoglobulin superfamily, is a multifunctional glycoprotein that has been shown to increase tumor invasion, metastasis and multidrug resistance. To define the role of CD147 in invasion and metastasis more precisely, we utilized gene silencing to inhibit the expression of CD147 in pancreatic cancer cells. We observed that CD147 expression was significantly impeded at both the mRNA and protein levels and resulted in a decrease of MMP-2 and MMP-9 activities. There was also a decrease of MCT1 expression in the invasion and metastasis potential of pancreatic cancer cells, as well as increased chemosensitivity to gemcitabine in Panc-1 cells. Overall, these results suggest that CD147 plays an important role in the invasion, metastasis and chemosensitivity of the human pancreatic cancer cell line Panc-1, indicating that CD147 may be a promising therapeutic target for pancreatic cancer.
ONCOLOGY REPORTS 27: 2003-2009, 2012
Abstract. CD147, which belongs to the immunoglobulin
superfamily, is a multifunctional glycoprotein that has been
shown to increase tumor invasion, metastasis and multidrug
resistance. To dene the role of CD147 in invasion and metas-
tasis more precisely, we utilized gene silencing to inhibit the
expression of CD147 in pancreatic cancer cells. We observed
that CD147 expression was significantly impeded at both
the mRNA and protein levels and resulted in a decrease of
MMP-2 and MMP-9 activities. There was also a decrease of
MCT1 expression in the invasion and metastasis potential of
pancreatic cancer cells, as well as increased chemosensitivity
to gemcitabine in Panc-1 cells. Overall, these results suggest
that CD147 plays an important role in the invasion, metastasis
and chemosensitivity of the human pancreatic cancer cell line
Panc-1, indicating that CD147 may be a promising therapeutic
target for pancreatic cancer.
Pancreatic cancer is a malignancy with an extremely poor
prognosis and is refractory to conventional chemotherapy
and radiotherapy. Despite efforts in the past years, conven-
tional treatment approaches, such as surgery, radiation,
chemotherapy, or combinations of these, the mortality rate of
pancreatic cancer still remains high (1-4). Therefore, it can be
effectively diagnosed, prevented, and treated only by devel-
oping a detailed understanding of the molecular biology of
underlying pancreatic cancer formation and progression.
CD147 (EMMPRIN) is a highly glycosylated cell surface
transmembrane protein that belongs to the immunoglobulin
superfamily (5), and it is thought to be involved in inamma-
tion, neural-glial interaction, and virus infection (6-9). CD147
is found to be highly expressed in a variety of malignant human
cancers, including malignancies of the pancreas (6,10,11), and
it induces tumor cell invasion by stimulating the production of
matrix metalloproteinases (MMPs), resulting in tumor inva-
sion and metastasis (12). In addition, CD147 plays a pivotal
role as a chaperone for the proper plasma membrane expres-
sion and the activity of monocarboxylate transporters (MCTs),
particularly MCT1 and MCT4 (13-15). MCTs are among
the most important cellular pH regulators likely involved in
cancer pH homeostasis (16-18). The MCT family has fourteen
members (19), six of which have been functionally character-
ized, but only MCT1-MCT4 have been shown to catalyze the
proton-coupled transport of lactate (20-24). Many studies
have demonstrated that CD147 acts as an essential chaperone
to take MCT1 and MCT4 to the plasma membrane where the
MCTs and CD147 are tightly associated (13,25).
CD147 is also involved in multidrug resistance of cancer
cells via hyaluronan-mediated activation of ErbB2 signaling
and cell survival pathway activities, but the mechanism of
CD147 in multidrug resistance of pancreatic cancer remains
elusive (26-28). We demonstrate here that CD147 silencing
inhibits pancreatic cancer cell invasion and metastasis and
increases chemosensitivity to gemcitabine. Our results
support the concept that CD147 expression is associated with
the malignant potential of cancer cells, since it sustains the
expression and function of MMPs and MCTs.
Materials and methods
Plasmid constructs and generation of stable cell clones. The
vector pSilencer 3.1-H1 neo (Ambion) was used to generate
short hairpin RNA (shRNA) specically for CD147. Two pairs
CD147 silencing via RNA interference reduces
tumor cell invasion, metastasis and increases
chemosensitivity in pancreatic cancer cells
YUQIN PAN1*, BANGSHUN HE1*, GUOQI SONG1, QIAN BAO2,
ZHIPENG TANG1, FULIANG TIAN2 and SHUKUI WANG1
1Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu 210006;
2Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210046, P.R. China
Received February 1, 2012; Accepted March 5, 2012
Correspondence to: Dr Shukui Wang, Nanjing First Hospital,
Nanjing Medical University, Nanjing, Jiangsu 210006, P.R. China
Abbreviations: MMP, matrix metalloproteinase; MCT, mono-
carboxylate transporter; LDH, lactate dehydrogenase; shRNA, short
hairpin RNA; PPC, plasma peak concentrations
Key word s: CD147, invasion, metastasis, multidrug resistance,
pancreatic cancer, gemcitabine
PAN et al: TARGETED GENE THERA PY BY CD47 SILENCING
of template oligonucleotides, each encoding one of the target
sequences, were designed and synthesized (designated as
shRNA1 and shRNA2, respectively), and the scrambled control
sequence (designated as shRNA-control) was also synthesized
(Table I). Subsequently, these oligonucleotides were cloned
into the plasmid pSilencer 3.1-H1 neo, using restriction endo-
nuclease BamHI and HindIII. These recombinant vectors
were designated as pH1-shRNA-control, pH1-shRNA1 and
pH1-shRNA2, respectively. The product was conrmed by
DNA sequenci ng.
The plasmids carrying the short hairpin RNA were
transfected into pancreatic cancer cells, using liposome
Lipofectamine 2000 (Invitrogen-Life Technologies, Carlsbad,
CA, USA), and subsequently cultured in the presence of
500 µg/ml G418 (Gibco-BRL, Grand Island, NY, USA) for
Quantitative real-time PCR assays. Total RNA was extracted
with TRIzol (Invitrogen-Life Technologies), according to
the manufacturer's instructions. Following treatment with
DNase I (Takara Biotechnology Co., Ltd., Dalian, China) at
37˚C for 30 min, RNA quantication was performed using
spectrophotometry. The primers used for CD147, MCT1,
MCT4 and β-actin are listed in Table II. The mRNA levels
for CD147, MCT1, MCT4 and β-actin were analyzed by a one-
step real-time reverse transcriptase polymerase chain reaction
with RNA-direct™ SYBR-Green Real-time PCR Master Mix
(Toyobo Co., Ltd., Osaka, Japan), according to the manufac-
turer's instructions. The cycling conditions were as follows: 40
cycles consisting of denaturation at 95˚C for 5 sec, annealing at
60˚C for 5 sec, and extension at 72˚C for 30 sec. The Ct used in
the real-time PCR quantication was dened as the PCR cycle
number that crossed an arbitrarily chosen signal threshold in
the log phase of the amplication curve. To verify the fold
change of target gene expression, calculated Ct values were
normalized to Ct values of β-actin amplied from the same
sample (ΔCt = Ctt arget - Ctβ-act in), and the 2-ΔΔCt method was used
to calculate fold change (29). Each sample was prepared in
triplicate, and all reactions were triplicated independently to
ensure the reproducibility of the results.
Western blot analysis. The expression of CD147, MCT1
and MCT4 protein was evaluated by western blot analysis.
Total protein was separated by SDS-PAGE on 12% gels and
transferred to a polyvinylidene diuoride (PVDF) membrane.
Skim milk powder (5%) (soluble in TBST buffer solution) was
used at room temperature under sealed conditions for 1 h,
with mouse anti-CD147 primary antibodies (1:500), rabbit
anti-MCT1 primary antibodies (1:500), rabbit anti-MCT4
primary antibodies (1:500) and rabbit anti-human β-actin
primary antibodies (1:500) incubated at room temperature for
2 h, followed by incubation in a 1:2000 dilution of secondary
antibodies conjugated to horseradish peroxidase (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) for 1 h at room temper-
ature. The protein was visualized by ECL. All of the western
blot analyses were performed at least three times.
Determination of intracellular lactate concentration. The
change of intracellular lactate concentration in Panc-1 cells
after CD147 silencing was assessed using a lactic acid assay
kit (KeyGen Biotech Co., Ltd., Nanjing, China). This assay is
based on the catalysis of lactate dehydrogenation by lactate
dehydrogenase (LDH) to generate pyruvate by NAD+ as
hydrogen acceptors. Subsequently, nitroblue tetrazolium
(NBT) is reduced to pur ple coloring when hydrogen is delivered
to it from phenazine methosulphate (PMS). There is a linear
relationship between the absorbance at 530 nm and the lactate
concentration. Cells (1x106) were harvested by centrifugation,
and cells were then ruptured by hypotonic salt solution for
1 h at room temperature. The supernatant was retained after
centrifuging. The optical density was read at 530 nm. Graphs
are representative of three separate experiments.
In vitro invasion assay. Transwell plates (Corning Costar,
Cambridge, MA, USA) were coated with basement membrane
Matrigel (20 mg/ml, Becton-Dickinson, Franklin Lakes, NJ,
USA) for 4 h at 37˚C. After the Matrigel solidied, 1x105
Table I. Sequences of the designed CD147 specic shRNAs.
Table II. Primers of CD147, MCT1, MCT4 and β-actin for
CD147 Sense: 5'-CCATGCTGGTCTGCAAGTCAG-3'
MCT1 Sense: 5'-CACTTAAAATGCCACCAGCA-3'
MCT4 Sense: 5'-GTTGGGTTTGGCACTCAACT-3'
β-actin Sense: 5'-CTGGAACGGTGAAGGTGACA-3'
ONCOLOGY REPORTS 27: 2003-2009, 2012 2005
cells were seeded onto the Matrigel and incubated at 37˚C for
24 h. After 18 h, cells that migrated through the permeable
membrane were xed with 100% methanol for 10 min. The
membrane with cells were soaked in 0.1% crystal violet for
10 min and then washed with distilled water. The number of
cells attached to the lower surface of the polycarbonate lter
was counted at x400 magnication under light microscopy.
Each assay was carried out in triplicate and repeated three
Drug sensitivity assay. To assess their multidrug chemo-
sensitivity, cells were plated in 96-well plates at a density of
1x104 cells/well and further incubated for 24 h. The medium
was then removed and replaced with fresh medium containing
gemcitabine, paclitaxel, and oxaliplatin, respectively, with
varying PPC (plasma peak concentrations, 0.1 PPC, 1.0 PPC
and 10.0 PPC) for another 48 h. After that, cells were stained
with 20 µl of sterile MTT dye [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, 5 mg/ml; Sigma] at 37˚C
for 4 h, followed by removing the culture medium and mixing
150 µl of dimethylsulfoxide (DMSO) thoroughly for 10 min.
Spectrometric absorbance at 490 nm was measured with a
microplate reader. Each group was plated in three wells and
was repeated three times.
In vivo metastasis assay. We used 4-6-week-old male BALB/c
nude mice (Center for Comparative Medical Research of the
Yangzhou University, Yangzhou, China). Cells were washed
and resuspended in serum-free DMEM before inoculation.
In each of the nude mice (n=8), 2x105 cells in 200 µl culture
medium were inoculated into the tail vein. Six weeks after
inoculation, all animals were euthanized and the lungs were
removed. Harvested tissues were fixed in 10% buffered
formalin, embedded in parafn, sectioned at 4 µm, and stained
with H&E. The antitumor effect was evaluated by counting the
number of metastatic tumor clones on the surface of the lungs.
All experiments were performed in accordance with institu-
tional guidelines for the care and use of experimental animals.
Statistical analysis. Statistics were conducted by the SPSS
software. Experimental data are presented as the mean ± SD
(standard deviation) and assessed by Student's t-tests and
one-way ANOVA at a signicance level of P<0.05.
shRNA targeting CD147 suppresses CD147 expression in
Panc-1 cells. To better understand the role of CD147 in tumor
cells, we established two recombinant vectors, including
pH1-shRNA1 and pH1-shRNA2. As demonstrated by quan-
titative reverse transcription PCR (qRT-PCR), pH1-shRNA2
effectively inhibited expression of CD147 in tumor cells
(P< 0.01) (Fig. 1A). In addition, western blot analysis conrmed
the downregulation of CD147 protein by pH1-shRNA2
(P<0.01) (Fig. 1D).
CD147 silencing inhibits MCT1 and MCT4 expression. Many
studies have demonstrated that the functionality of MCT1
and MCT4, natural transporters of lactate, on mitochondrial
membranes depends on the association with the mature,
Figure 1. Expression levels of CD147, MCT1 and MCT4 in Panc-1 cells after CD147 silencing. Relative mRNA levels of (A) CD147 (B) MCT1 (C) MCT4
were analyzed by quantitative RT-PCR. β-actin was used as the norma lization control. *P<0.01 compared with the control group. Graphs are representative
of three separate exper iments. (D) Western blot analysis of CD147, MCT1 and MCT4 protein expression levels. β-actin was used as the loading control. The
results show that the protein expression levels of CD147 and MCT1 were signicantly downregulated by pH1-shRNA2 in Panc-1 cells. There was no signicant
change of MCT4 protein levels. The data were obtained from three independent experiments.
PAN et al: TARGETED GENE THERA PY BY CD47 SILENCING
glycosylated form of CD147. We thus examined whether
CD147 silencing could reduce the expression of MCT1 and
MCT4. As demonstrated by qRT-PCR, the mRNA expression
of MCT1 was downregulated by pH1-shRNA2 in the Panc-1
cell line (P<0.01), but the expression of MCT4 was not signi-
cantly altered (P>0.05) (Fig. 1B and C). In addition, western
blot analysis conrmed the downregulation of MCT1 protein
by pH1-shRNA2 in the Panc-1 cell line. Furthermore, there
was no signicant change of the MCT4 protein (Fig. 1D).
CD147 silencing reduces MMP-2 and MMP-9 activities.
CD147 has been suggested to induce MMP in tumor-associated
mesenchymal cells, so we examined whether CD147 silencing
could reduce the activities of MMP-2 and MMP-9, using
gelatin zymography. The activities of MMP-2 and MMP-9
were reduced signicantly by pH1-shRNA2 in the Panc-1 cell
line, compared with the control group (P<0.01); and there was
no significant difference between the pH1-shRNA-control
group and the corresponding controls (P>0.05) (Fig. 2).
CD147 silencing inhibits the function of lactate transporters.
We then examined whether CD147 silencing inhibited the func-
tion of lactate transporters. To conrm that the downregulation
of MCT1 expression by pH1-shRNA2 inhibits the function of
these transporters we assessed the intracellular lactate concen-
tration in Panc-1 cells. As shown in Fig. 3, the CD147 silencing
induced an increase of the intracellular lactate concentration
in Panc-1 cells (P<0.01). This demonstrates that the CD147
silencing-induced decrease in MCT1 expression is associated
with an increase in intracellular lactate concentration.
Inhibition of CD147 alters tumor cell invasion in vitro. To
examine whether the downregulation of CD147 in Panc-1
cells affected its invasive ability, we performed an in vitro
Matrigel transwell analysis. The results showed that silencing
of CD147 signicantly reduced invasion activities in Panc-1
cells when compared with corresponding controls (P<0.05)
CD147 silencing increases the sensitivity to chemotherapeutic
drugs. CD147 has been found to be overexpressed in multidrug
resistance tumor cells and could confer resistance to some
antitumor drugs. To examine whether the downregulation of
CD147 in Panc-1 cells affected its sensitivity to chemothera-
peutic drugs, we assessed whether CD147 silencing induced
an alteration in the chemosensitivity of Panc-1 cells to various
agents by the MTT assay. As shown in Fig. 5, CD147 silencing
signicantly increased the chemosensitivity of Panc-1 cells
to gemcitabine at 1.0 PPC and 10.0 PPC but not at 0.1 PPC
compared with the control groups (P<0.05). The results also
show that there was no signicant change of the chemosensi-
tivity induced by CD147 silencing to paclitaxel and oxaliplatin
in Panc-1 cells (P>0.05).
CD147 silencing inhibited the metastatic potential of Panc-1
cells. To investigate the effect of CD147 silencing on Panc-1-
cell metastasis, we injected cells (2x105⁄200 µl) into the tail
vein of nude mice and evaluated the presence of metastatic
nodes in the lungs after 6 weeks. We observed a signicant
reduction in the number of metastatic nodes in the mice
that received Panc-1 cells stably transfected with CD147, as
compared with the corresponding controls (P<0.01) (Fig. 6).
Figure 2. Activities of MMP-2 and MM P-9 in Panc-1 cells after CD147
silencing. Cells were incubated for 24 h and conditioned media were used for
the measur ement of MMP-2 and MMP-9 protein levels by gelatin zy mography.
(A) Photographs of the MMP-2 and MMP-9 bands, which are representative
of three independent experiments. Quantitative analysis of the ( B) MMP-2
bands and (C) the MMP-9 bands. *P<0.01 compared with the control. #P<0.01
compared with the control. Graphs are representative of three separate experi-
Figure 3. Intracellular lact ate in Panc-1 cells after CD147 silencing. Panc-1
cells were transfected with pH1-shRNA2-CD147. CD147 silencing increased
the intracellular lactate concentration (P<0.01). Transfection with pH1-
shRNA2- control ser ved as a negative control. The graph is representative of
three separate experiments.
ONCOLOGY REPORTS 27: 2003-2009, 2012 2007
CD147 is a multifunctional glycoprotein that has been shown
to increase tumor invasion. It plays an important role in cancer
progression, such as promoting invasiveness via the stimulation
of matrix metalloproteinase production (MMPs), interacting
with certain lactate transporters (MCT1 and MCT4) and
facilitating their expression on the cell surface, and mediating
multidrug resistance via the hyaluronan-mediated upregula-
tion of ErbB2 signaling and cell survival pathway activities
(13,26,30). CD147 is highly expressed on the surface of various
tumors, including pancreatic cancer (31,32); however, the
molecular mechanisms involved and the role of CD147 in
pancreatic cancer remain poorly understood. In the present
study, we constructed the CD147 shRNA expression vector to
inhibit the expression of CD147 in the pancreatic cancer cell
Figure 4. Invasive ability of Panc-1 cells after CD147 silencing. Using a
Matrigel polycarbonate lter, 1x105 cells were seeded in the Millicell upper
chamber (A) Representative images of cells invading the Matrigel (x400). (B)
The number of cells that invaded through the chamber was averaged. The
invading cells were counted as a sum of 10 elds of vision under a micro-
scope. *P<0.01 compared with control group. The graph is representative of
three separate experiments.
Figure 5. Multidrug chemosensitivity of Panc-1 cells after CD147 silencing. (A) Cells were treated with gemcitabine with var ying plasma peak concentrations
(PPC; 0.1, 1.0 and 10.0 PPC) for 48 h. Cell viability was determined by the MTT assay. CD147 silencing signicantly increased the chemosensitivity of Panc-1
cells to gemcitabine at 1.0 PPC and 10.0 PPC compared with the control groups (*P<0.05, #P<0.05). The graphs are representative of three separate experi-
ments. Cells were treated with (B) paclitaxel or (C) oxaliplatin with var ying PPC (0.1, 1.0 and 10.0 PPC) for 48 h. Cell viability was determined by the MTT
assay. The results show that there was no signicant change of the chemosensitivity induced by CD147 silencing to paclitaxel or oxaliplatin in Panc-1 cells
(P>0.05). Graphs are representative of three separate experiments.
PAN et al: TARGETED GENE THERA PY BY CD47 SILENCING
line in order to investigate the role of CD147 silencing in inva-
sion, metastasis, and multidrug resistance of pancreatic cancer.
Tumor cell invasion and metastasis are the main causes
of treatment failure and mortality in patients. CD147 can
stimulate the production of MMPs, thereby leading to extra-
cellular matrix degradation and increased tumor invasion and
metastasis. It has been reported that the expression of MMP-2
and MMP-9 is correlated with the invasion and local recur-
rence rate in pancreatic cancer cells (33-35). Transfection of
CD147 cDNA into human MDA-MB-436 breast cancer cells
resulted in an enhancement of tumor growth and an increase in
metastatic incidences, both of which were directly correlated
with high levels of tumor-derived MMP-2 and MMP-9 (36).
In the present study, the results showed that CD147 silencing
in human pancreatic cancer cells reduced the secretion of
MMP-2 and MMP-9 and inhibited the invasion and metastasis
ability of pancreatic cancer cells in vitro. This was consistent
with previous studies (33-35).
CD147, by its close association with MCT1 and MCT4, plays
a pivotal role in the glycolysis reected by the transmembrane
transport of lactate and the regulation of cell proliferation.
Tumor cell expression of MCT1 and MCT4 has been reported
to be regulated by CD147, which facilitates their cell surface
expression, so CD147 plays a pivotal role in the glycolysis
reected by the transmembrane transport of lactate and the
regulation of cell proliferation (37). MCT1 inhibition has also
been shown to have antitumor potential against in vivo models
of lung carcinoma, colorectal carcinoma, and a squamous
carcinoma cell line after a cyano-4-hydroxycinnamate-medi-
ated MCT1 inhibition (38). The present results showed that
CD147 silencing resulted in a signicant reduction of MCT1,
but not MCT4 expression, which supports the concept that
CD147 is an ancillary protein required for the expression of
MCT1. The results also showed that CD147 silencing resulted
in an increase of the intracellular lactate concentration in
pancreatic cancer cells. The increase of lactate concentration
may inhibit the cell growth, since lactate has been demon-
strated to decrease pyruvate reduction to lactate by inhibition
of (LDH) (39). However, the present results did not show that
the increase of lactate concentration inhibited the proliferation
in Panc-1 cells (data not shown), and we will investigate the
mechanism in the future.
Multidrug resistance is a major obstacle in the treatment
of pancreatic cancer, and upregulation of CD147 has been
reported in multidrug resistant cancer cells. The relationship
between tumor metastasis and multidr ug resistance is not ful ly
dened in pancreatic cancer, although indirect evidence in
the advanced disease suggests a functional link between these
processes. In the present study, CD147 silencing increased
the chemosensitivity to gemcitabine, but not to paclitaxel and
oxaliplatin in Panc-1 cells, suggesting that the expression of
CD147 is closely related to multidrug resistance in pancreatic
cancer. Gemcitabine is the rst-line chemotherapeutic agent
for advanced adenocarcinoma of pancreatic cancer; however,
chemoresistance to gemcitabine remains a major cause of
failure for the clinical treatment of this disease. Studies
have indicated that resistance to gemcitabine is dependent
on mitochondria-mediated apoptosis, but various mediators
of gemcitabine-mediated apoptosis have been described
(40-43). The precise mechanism is not fully understood.
Collectively, these observations identify CD147 as a key
regulator of the invasion, metastasis, and multidrug resistance
in pancreatic cancer cells and suggest that patients with this
malignancy may benefit from targeted therapies blocking
effectors of this signaling pathway.
This project was supported by grants from the National Nature
Science Foundation of China (no. 81172141), Nanjing Science and
Technology Committee project (no. 201108025), and Nanjing
Medical Technology Development Project (no. ZKX11025).
Figure 6. CD147 silencing inhibits the metastatic nodes in the lungs. A
volume of 2x105 cells⁄ 200 µl was injected into each of the nude mice through
the tail vein. (A) Microscope images show that CD147 silencing signicantly
inhibits the lung metastatic nodes after inoculation with Panc-1 cells (H&E
staining). Magnication, x200. (B) The number of lung metastatic nodes.
*P<0.01 compared with the control group.
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