Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells
et al. (2013) Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In
Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells. PLoS ONE 8(8): e67268. doi:10.1371/journal.pone.0067268
Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells
Wei Zhang 0
Pu Zhao 0
Xiu-Li Xu 0
Lei Cai 0
Zhen-Shun Song 0
Da-Yong Cao 0
Kai-Shan Tao 0
Wen- Ping Zhou 0
Zhi-Nan Chen 0
Ke-Feng Dou 0
Jung Weon Lee, Seoul National University, Korea, Republic of
0 1 Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University , Xi'an, Shaanxi Province , China , 2 Department of Hepatobiliary Surgery, General Hospital of Shenyang Military Area Command , Shenyang, Liaoning Province , China , 3 College of Life and Health Sciences, Northeastern University , Shenyang, Liaoning Province , China , 4 Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer Biology, State Key Discipline of Cell University, Fourth Military Medical University , Xi'an, Shaanxi Province , China , 5 Center of Clinical Laboratory Medicine of People's Liberation Army, Xijing Hospital, Fourth Military Medical University , Xi'an, Shaanxi Province , China
It has been reported that Annexin A2 (ANXA2) is up-regulated in hepatocellular carcinoma (HCC), but the roles of ANXA2 in the migration and invasion of HCC cells have not been determined. In this study, we found that ANXA2-specific siRNA (siANXA2) significantly inhibited the migration and invasion of HCC cells co-cultured with fibroblasts in vitro. In addition, the production of MMP-2 by fibroblasts cultured in supernatant collected from si-ANXA2-transfected HCC cells was notably down-regulated. ANXA2 was also found to be co-localized and co-immunoprecipitated with CD147. Further investigation revealed that the expression of ANXA2 in HCC cells affected the shedding of CD147-harboring membrane microvesicles, acting as a vehicle for CD147 in tumor-stromal interactions and thereby regulating the production of MMP-2 by fibroblasts. Together, these results suggest that ANXA2 enhances the migration and invasion potential of HCC cells in vitro by regulating the trafficking of CD147-harboring membrane microvesicles.
Funding: This work was supported by grant 20121086 from Dr. Start-up Fund of Liaoning Province, China. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The annexins are a family of Ca2+-dependent
phospholipidbinding proteins with various membrane-related functions. The
name annexin is derived from the Greek annex, which means
bring/hold together, and was chosen to describe the principal
property of all, or at least nearly all, annexins the binding to
and possibly holding together of certain biological structures, in
particular membranes . At least 20 members of the family have
been described to date . Annexin A2 (ANXA2), also called
Annexin II, is one of the best characterized of the Annexins.
ANXA2 is composed of two main structural domains: the 33-kDa
C-terminal conserved core domain, which contains the Ca2+- and
membrane-binding sites [1,3]; and the 3-kDa N-terminal variable
domain, which contains the protein binding sites and
phosphorylation sites. Otherwise, the N-terminus harbors a high affinity
hydrophobic interaction site for the EF-hand Ca2+ binding protein
S100A10 (p11). Two molecules of ANXA2 and two molecules of
p11 form a heterotetrameric complex (A2t) that has been
suggested to be involved in exocytosis, endocytosis and membrane
vesicle trafficking . ANXA2 was first discovered as a substrate
of the Rous sarcoma virus-encoded tyrosine protein kinase.
Subsequent studies have implicated ANXA2 in several biological
functions including mitogenic signal transduction , fibrinolysis
, immune response , proliferation , carcinogenesis and
tumor progression [6,9,1114]. Large-scale genomic and
proteomic studies have begun to accumulate evidence regarding the
association and possible involvement of ANXA2 with benign and
malignant neoplasms of diverse origins . Increased expression
of ANXA2 has been described in a large number of spontaneous
neoplasms, including pancreatic cancer, gastric carcinoma,
colorectal cancer, breast cancer, high-grade gliomas and kidney
cancer (reviewed in ) and is positively correlated with tumor
invasion and migration . In contrast, the expression of
ANXA2 is lost or reduced in prostate cancer, and the role of
ANXA2 in prostate cancer appears contradictory [17,18]. The
differential expression of ANXA2 in HCC and normal liver tissue
has been reported, but a more detailed functional assessment is
Although published data support a crucial role for ANXA2 in
tumor progression, the detailed mechanisms underlying this role
have yet to be fully elucidated. Breakdown of the extracellular
matrix (ECM), which is mediated by a variety of proteases, endows
malignant cells with the ability to penetrate through tissue barriers
and is believed to play a major role in tumor migration and
invasion. ANXA2 has been found to be a putative co-receptor for
both plasminogen and tissue-type plasminogen activator (tPA)
. Cell surface ANXA2 acts as a platform for plasmin
activation, where inactive plasminogen is cleaved by tPA to yield
the active serine proteinase, plasmin, thereby facilitating the
migration and invasion of malignancies. Studies have also
demonstrated that ANXA2 may regulate the production and
activation of matrix metalloproteinases (MMPs) [20,21].
CD147 is a widely distributed cell surface glycoprotein that
belongs to the immunoglobulin superfamily. It was first identified
as a factor shedding from the surface of tumor cells that is
responsible for stimulating the production of MMP-1 by
fibroblasts . Accumulating evidence indicates that CD147 is
a major mediator of the malignant phenotypes of various tumors
. CD147 induces angiogenesis by stimulating the production
of VEGF, invasiveness by stimulating the production of MMPs
and multidrug resistance via hyaluronan-mediated up-regulation
of ErbB2 signaling and the activity of cell survival pathways .
Induction of MMP production through cell interactions is one of
the most important functions of CD147 thus the derivation of
its other name: extracellular matrix metalloproteinase inducer
(EMMPRIN) . CD147 may serve as its own counter-receptor
in homotypic cancer cell interactions and cancer cell-fibroblast
interactions, thereby stimulating the production of MMPs via a
homophilic interaction with other CD147 proteins [26,27]. In
addition, MT1-MMP, MMP-2, and MMP-9 have been reported
to cleave and release a shorter form of soluble CD147 that lacks
the C-terminus, thereby modulating the expression of MMPs
[26,28]. Interestingly, recent studies have provided evidence that
membrane microvesicles shed from tumor cells carry full-length
CD147 and play a role in tumorstromal interactions through the
upregulation of the production of MMPs [29,30]. Previous studies
have demonstrated that CD147 promotes the invasion and
metastasis of human hepatoma cells by stimulating both tumor
cells and peritumoral fibroblasts to produce elevated levels of
MMPs, although the modulation of fibroblasts is the more critical
part of the process [31,32].
Although the overexpression of ANXA2 in HCC has been
shown, the role of ANXA2 in the migration and invasion of HCC
cells remains obscure [33,34]. In the present study, we knocked
down the expression of ANXA2 in HCC cells to explore its role in
HCC cell migration and invasion. To further investigate the
mechanisms of ANXA2 in tumor progression, we introduced
CD147, which has been hypothesized to interact with ANXA2 but
which has not yet been shown to do so . Based on the
involvement of ANXA2 in exocytosis and membrane vesicle
trafficking [4,5] and the role of CD147-harboring membrane
microvesicles on tumor progression, we hypothesized that ANXA2
is involved in the shedding of CD147-harboring microvesicles
from tumor cells, thereby regulating tumor migration and
Materials and Methods
Two highly invasive human HCC cell lines, SMMC-7721 and
FHCC-98, were cultured in DMEM containing 10% fetal bovine
serum (FBS). SMMC-7721 cells were obtained from the Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Science . The FHCC-98 cells were purchased from the Cell
Engineering Research Centre, Fourth Military Medical
University, China . Human embryo pulmonary fibroblast-1 (HPF-1)
cells were purchased from the Chinese Academy of Medical
Sciences and were cultured as described above .
si-ANXA2 was purchased from Santa Cruz Biotechnology, Inc.
(sc-29199) . si-CD147
(59-GUUCUUCGUGAGUUCCUCtt39, 39-dTdTCAAGAAGCACUCAAGGAG-59) was synthesized
by Ambion, Inc . HCC cells were transfected with siRNA
using LipofectAMINE 2000 according to the manufacturers
instructions (Invitrogen, USA). Silencer negative control siRNA
(snc-RNA) (Ambion, USA) was used as a negative control under
Forty-eight hours after transfection with siRNA, total RNA was
extracted from the cells with TRIzol (Invitrogen, USA) and reverse
transcribed into cDNA using a ReverTra Ace-a-TM kit
(TOYOBO, Japan). GAPDH was used as an internal control.
All primers were synthesized by Shanghai Sangon Co. as follows:
ANXA2, forward primer
59-GAGGATGGCTCTGTCATTGATT-39; reverse primer
59-ACCACAGTCCATGCCATCAC-39 and 59-TCCACCACCCTGTTGCTGTA-39.The
conditions for PCR were one cycle at 94uC for 4 min, 40 cycles
at 94uC for 30 s, 60uC for 30 s, and 72uC for 30 s. PCR products
were electrophoresed on 1% agarose gels. All PCR reactions were
performed in triplicate.
Cells were harvested and lysed in lysis buffer. A BCA Protein
Assay Kit (Pierce Biotechnology, USA) was employed to determine
the concentration of total protein. Equal amounts of protein were
separated by SDS-PAGE (12%). Proteins were transferred to a
polyvinylidene fluoride (PVDF) microporous membrane
(Millipore, USA) and the blots probed with ANXA2 mAb (Santa Cruz,
USA) or CD147 mAb (Santa Cruz, USA). Tubulin was chosen as
an internal control, and the blots were probed with mouse
antitubulin mAb (Santa Cruz, USA).
In vitro migration/invasion assays
The assays were performed using chambers with polycarbonate
filters (pore size, 8 mm). Some filters were coated with Matrigel
(Becton Dickinson Labware, USA), and some were not.
Twentyfour hours after being transfected with siRNA, HCC cells were
harvested. Equal numbers (56104) of transfected and HPF-1 cells
were then placed in the upper chamber in 300 mL of 0.1% serum
medium. Cells transfected with snc-RNA were used as a negative
control. The lower chamber was filled with 0.1% fetal bovine
serum medium (200 mL) and serum-free conditioned medium
collected from HPF-1 cells (200 mL). After 8 h (migration assay) or
24 h (invasion assay) of incubation, the cells in the upper chamber
were carefully removed with a cotton swab. The cells on the
underside were then fixed in methanol, stained with H&E, and
counted under a microscope.
Serum-free conditioned medium collected from
siRNA-transfected SMMC-7721 and FHCC-98 cells was added to HPF-1 cells.
Fifteen hours later, the conditioned medium was collected and
separated using a 10% acrylamide gel containing 0.1% gelatin.
The gels were incubated in a 2.5% Triton X-100 solution at room
temperature with gentle agitation and were then soaked in
reaction buffer [0.05 mol/L Tris-HCl (pH 7.5), 0.2 mol/L NaCl,
and 0.01 mol/L CaCl2] at 37uC for 18 h. After the reaction, the
gels were stained for 6 h with Coomassie brilliant blue and
destained for 0.5 h. Areas of gelatinolytic activity were evident based
on their negative staining.
Immunocytochemistry and confocal microscopy
SMMC-7721 and FHCC-98 cells were harvested and allowed
to attach to pre-coated glass coverslips for 24 hours. They were
then fixed in 3.7% formaldehyde in PBS, permeabilized with 0.1%
Triton X-100 and blocked with 1% BSA (Fraction V) in PBS for
1 h. Coverslips were incubated with goat anti-CD147 antibody
(1:100; Santa Cruz, USA) and ANXA2 mAb (1:100) in PBS
overnight at 4uC. The cells were then washed and incubated with
Alexa 594-conjugated goat anti-mouse (Invitrogen, USA) or
donkey anti-goat IgG-FITC (Santa Cruz, USA) secondary
antibody at a dilution of 1:400 for 1 h at room temperature. Cell
nuclei were stained with DAPI (Vector, USA). The slides were
then washed and mounted onto glass slides. Anti-fade was added
to prevent quenching of the fluorophores. The proteins were
visualized with a FluoViewTM FV1000 confocal microscope
Extraction and co-immunoprecipitation of total cellular
Total cellular membrane proteins (TMP) were extracted from
SMMC-7721 and FHCC-98 cells using a Plasma Membrane
Protein Extraction Kit (BioVision, USA), according to the
manufacturers instructions. SMMC-7721 and FHCC-98 cells
(56107) were collected, centrifuged and washed with 1 ml ice-cold
PBS. The cells were then re-suspended in 1 ml Homogenize
Buffer Mix in an ice-cold Dounce homogenizer 3050 times. The
homogenate was centrifuged at 700 g for 10 minutes at 4uC. The
supernatant was collected and centrifuged at 10,000 g for
30 minutes at 4uC. The cytosol fraction (supernatant) and total
cellular membrane proteins (pellet) were collected. The samples
were either used immediately or stored at 220uC for further study.
The interaction of ANXA2 with CD147 in the plasma
membranes of SMMC-7721 and FHCC-98 cells was detected
using a ProFoundTM Mammalian Co-Immunoprecipitation Kit
(Pierce, USA) according to the manufacturers instructions. Briefly,
TMP extracted as described above were collected onto a Coupling
gel pre-bound with 200 mg anti-ANXA2 mAb, anti-human
CD147 mAb, or anti-JEV mAb (mouse IgG, kindly provided by
the Department of Microorganism, Fourth Military Medical
University and used as a negative control), followed by four
washes with the co-immunoprecipitation buffer. The coupling gel
was washed with elution buffer, and aliquots of the eluent were
used for Western blotting with anti-ANXA2 mAb and anti-CD147
Isolation and electron microscopy of membrane
Microvesicles were isolated as previously described . Briefly,
medium conditioned for 24 hours by subconfluent, healthy
SMMC-7721 cells was centrifuged at 600 g for 15 minutes and
then at 1500 g for 15 minutes to remove cells and large debris.
The supernatant was ultracentrifuged at 100,000 g for 1 hour at
4uC. Pelleted microvesicles were re-suspended in PBS (pH 7.4).
Isolated vesicles were quantified based on protein concentration
measurements using the Bradford method (Bio-Rad, Milan, Italy),
with BSA (Sigma, St. Louis, MO) used as a standard. Samples
were either frozen at 220uC or used immediately for electron
microscopy studies. Aliquots of vesicles were applied to 200 mesh
nickel parlodion-coated grids and allowed to settle. Samples were
negatively stained with 1% phosphotungstic acid (pH 6) before
being analyzed with an electron microscope .
Specific siRNA effectively down-regulates ANXA2
expression in HCC cells
To investigate the role of ANXA2 in the invasion and migration
of HCC cells, RNA interference was used to downregulate the
expression of ANXA2 in SMMC-7721 and FHCC-98 cells. Both
cell lines were transfected with si-ANXA2 and snc-RNA.
Fortyeight hours after transfection, the expression of ANXA2 mRNA
and protein was examined using RT-PCR and Western blot,
respectively. RT-PCR showed that si-ANXA2 could effectively
downregulate the expression of ANXA2 mRNA in SMMC-7721
and FHCC-98 cells, with an inhibition rate of 63.3963.87% and
62.8062.98%, respectively, compared to snc-RNA (p,0.001,
Fig. 1A). These results were consistent with Western blot assays.
The protein expression of ANXA2 was notably reduced in
siANXA2 transfected cells, with an inhibition rate of 64.1261.46%
and 58.8061.36%, respectively, compared to
To mimic in vivo tumor-stroma interactions within local
microenvironments, HCC cells were transfected with si-ANXA2
and then co-cultured with an equal number of HPF-1 cells in the
upper chamber. The migration assay showed that the number of
cells that migrated through the filter was drastically reduced in the
SMMC-7721 and FHCC-98 cells transfected with si-ANXA2,
with an inhibition rate of 59.2262.43% and 55.9462.76%,
respectively, compared to snc-RNA-transfected cells (p,0.001,
Fig. 2A). Using an in vitro invasion assay, the number of invading
cells was also shown to be reduced in si-ANXA2-transfected
SMMC-7721 and FHCC-98 cells, with an inhibition rate of
60.6361.65% and 59.4963.19%, respectively, compared to
sncRNA-transfected cells (p,0.001, Fig. 2B).
Both in vitro and in vivo, increased tumor aggression has been
reported to be correlated with high expression levels of MMPs,
which are mainly secreted by stromal cells. A critical question that
arises from our study is whether the expression level of ANXA2 in
HCC cells affects the production of MMPs by peritumoral
fibroblasts. To address this issue, we performed a gelatin
zymography assay, which indicated that the secretion of MMP-2
was significantly reduced in HPF-1 cells cultured in supernatant
collected from si-ANXA2-transfected HCC cells, with an
inhibition rate of 46.7563.29% and 35.6062.88% in SMMC-7721 cells
and FHCC-98 cells, respectively, compared to
snc-RNA-transfected cells (p,0.01, Fig. 2C). These data demonstrate that
ANXA2 and CD147 co-localize on HCC membrane
To study the possible interaction between ANXA2 and CD147
and to explore the hypothesis that ANXA2 is involved in the
shedding of CD147-harboring microvesicles from tumor cells, we
performed immunofluorescent double-labeling on SMMC-7721
and FHCC-98 cell slides. As shown in Fig. 3A, ANXA2 (red) and
CD147 (green) were co-localized in both cell lines. We focused on
the strong double-staining of the membrane. To further confirm
the results and explore the possibility that ANXA2 is involved in
CD147 membrane microvesicle trafficking, we extracted the TMP
from SMMC-7721 and FHCC-98 cells. Cytoskeletal protein
(tubulin), membrane protein (VEGF-R2), and ANXA2 were
detected in whole cell lysates and TMP and cytosol fractions
using Western blot (Fig. 3B). We then performed
co-immunoprecipitation of the TMP extracted from HCC cells. These results
showed that ANXA2 and CD147 co-immunoprecipitated with
each other in the TMP extracted from both SMMC-7721 and
FHCC-98 cells (Fig. 3C), indicating that ANXA2 and CD147
colocalize on HCC membrane, and there may be interaction effects
due to membrane-associated events between the two molecules.
ANXA2 affects the invasiveness of tumor cells by
regulating the transportation of CD147-harboring
Data have shown that ANXA2 is involved in microvesicle
trafficking  and that CD147-harboring microvesicles play an
important role in tumor-stroma cross-talk by stimulating the
production of MMPs [29,30]. The co-localization of ANXA2 and
CD147 described above indicated that ANXA2 may be involved
in CD147 membrane microvesicle trafficking, and could therefore
affect the invasiveness of tumor cells. To address this issue, we
isolated microvesicles shed from SMMC-7721 cells and analyzed
the harvested pellet using Western blot. Electron microscopy of the
ultracentrifuged pellet revealed that SMMC-7721 cells shed
spherical or ellipsoid-shaped microvesicles (,200500 nm),
consistent with previous reports on plasma membrane-derived
microvesicles . A representative image is shown in Fig. 4A.
The microvesicles were then analyzed using Western blot, which
showed that the microvesicles shed from SMMC-7721 cells carried
both ANXA2 and CD147 (Fig. 4A).
To further investigate the effect of ANXA2 on the shedding of
CD147-harboring microvesicles, SMMC-7721 cells were
transfected with si-ANXA2 or si-CD147 and the microvesicles then
isolated and analyzed as described above. The results (Fig. 4B)
showed that the expression of both ANXA2 and CD147 in the
isolated microvesicles was downregulated when SMMC-7721 cells
were transfected with si-ANXA2, with an inhibition rate of
64.4963.56% and 36.1661.49%, respectively, compared to the
snc-RNA-transfected group (p,0.01). However, when
SMMC7721 cells were transfected with si-CD147, only the expression of
CD147 in the microvesicles was significantly downregulated, with
an inhibition rate of 54.7860.64% compared to the
snc-RNAtransfected group (p,0.01).
We then focused on the effect of the isolated microvesicles on
the secretion of MMPs by fibroblasts. HPF-1 cells were treated
with microvesicles isolated from untreated or si-RNA-transfected
(snc-RNA, si-ANXA2 or si-CD147) SMMC-7721 cells. A gelatin
zymography assay (Fig. 4C) indicated that microvesicles isolated
from si-ANXA2- or si-CD147-transfected SMMC-7721 cells were
less efficient in the induction of MMP-2 compared to microvesicles
isolated from untreated or snc-RNA-transfected SMMC-7721 cells
(p,0.01). These results suggest that ANXA2 is involved in the
trafficking of CD147-harboring microvesicles derived from tumor
cells, which regulates the production of MMP-2 by fibroblasts,
thereby facilitating the progression of HCC.
ANXA2 is thought to be involved in the transduction of cellular
signals associated with inflammation, differentiation and
proliferation . Emerging evidence indicates that changes in the
expression and/or subcellular localization of ANXA2 contribute
to the development and progression of a variety of malignancies.
Frohlich et al. reported that ANXA2 was up-regulated in HCC
. Recently, Mohammad et al. confirmed the upregulation of
ANXA2 in HCC and provided a more detailed description .
They reported that ANXA2 is almost undetectable in normal liver
and chronic hepatitis tissue, but the expression of ANXA2 is
abundant in non-tumorous cirrhotic tissue at both the
transcriptional and translational levels. Furthermore, the expression of
ANXA2 is greater in tumorous tissue than in non-tumorous
cirrhotic tissue. These data strongly indicate that ANXA2 may be
involved in the malignant transformation and progression of
In the present study, we explored the role of ANXA2 in the
invasion and migration of HCC cells. Two HCC cell lines,
SMMC-7721 and FHCC-98, were co-cultured with HPF-1 cells to
mimic the tumor-stroma interaction system in vitro. A migration
assay, invasion assay, and gelatin zymography assay were
employed. Our results show that knocking down the expression
of ANXA2 in HCC cells inhibits the migratory and invasive
potential of these tumor cells and significantly attenuates the
production of MMPs by fibroblasts, which were previously
reported to be involved in human hepatic tumorigenesis and
metastasis. All these findings suggest that ANXA2 may play an
important role in the progression of HCC, including migration,
invasion, and enzyme degradation.
Previous studies have illustrated that ANXA2 functions in the
invasion and migration of various tumors. However, the exact
molecular mechanisms underlying this function remain largely
unknown. Sharma and Sharma proposed a mechanistic cascade
for the role of AXNA2 in tumor progression. They suggested that
ANXA2 at the cell surface of tumor/endothelial cells provides for
the mechanical assembly of plasminogen activator and
plasminogen, which locally activates plasminogen to plasmin. Plasmin
then induces the degradation of ECM, which in turn facilitates
endothelial/tumor cell invasion and migration . In this study,
we found that the treatment of HCC cells with ANXA2-specific
siRNA significantly reduced the production of MMP-2 by HPF-1
cells cultured in supernatant collected from HCC cells, suggesting
that certain factors may exist in the supernatant that regulate the
production of MMPs by HPF-1 cells. Accordingly, we focused on
CD147, an important molecule responsible for stimulating the
production of several MMPs (MMP-1, MMP-2, MMP-3, MMP-9,
MMP-14, and MMP-15) by fibroblasts and endothelial cells .
Thus, a possible interaction between ANXA2 and CD147 was
considered. In this study, CD147 was found to co-localize with
ANXA2 in HCC cells. The two molecules were also found to
coimmunoprecipitate with each other in TMP extracted from
SMMC-7721 and FHCC-98 cells. These results demonstrate that
ANXA2 and CD147 are in close proximity, if not directly
associated, and most likely interact in HCC cells.
If we assume that CD147 is the bioactive factor hiding in the
supernatant, how and in what form is CD147 released by tumor
cells? Vesicle shedding has been observed in normal cells under
certain physiological conditions  and is present at much higher
rates in tumor cells . The shedding of tumor surface antigens
in membrane vesicles has been implicated as an important feature
of malignant transformation. It is likely that vesicle shedding and,
more importantly, the factors released by vesicle shedding, are
vital to tumor survival and growth because it is by the release of
such factors that tumors condition their microenvironment,
regulate metastasis and evade immune surveillance . Sidhu
provided evidence of a form of tumor-stromal interaction. He
showed that the degradation of the ECM by fibroblasts is
controlled by the microvesicular release of CD147 from
NCIH460 cells . In addition, ANXA2 has been shown to
participate in the aggregation and transportation of membrane
microvesicles . Our present data support the possibility of an
interaction between ANXA2 and CD147. We then hypothesized
that CD147 carried by membrane microvesicles may be the
soluble bioactive factor affecting the production of MMPs in the
supernatant collected from HCC cells and that ANXA2 may be
involved in the trafficking. Our subsequent study confirmed this
hypothesis. Membrane microvesicles were successfully isolated
from the supernatant of SMMC-7721 cells using
ultracentrifugation. Both CD147 and ANXA2 were detected in the isolated
microvesicles using Western blot. When HPF-1 cells were treated
with microvesicles, the production of MMP-2 was significantly
increased. To further investigate the effect of ANXA2 on the
shedding of CD147-harboring microvesicles, SMMC-7721 cells
were transfected with si-ANXA2 or si-CD147 and microvesicles
then isolated. These results indicate that the downregulation of
ANXA2 in tumor cells reduces the expression of CD147 in
isolated microvesicles, but the expression of ANXA2 in the
microvesicles is not affected when cells are transfected with
siCD147. It has been reported that ANXA2 is involved in exocytosis
and membrane vesicle trafficking [4,5], which may be the reason
why the down-regulation of ANXA2 affects CD147 protein levels.
There is no evidence that CD147 regulates the production and
release of microvesicles, which is consistent with our result that the
down-regulation of CD147 did not affect ANXA2 protein levels.
The induction effect of microvesicles on the production of MMP-2
was weakened when SMMC-7721 cells were transfected with
either si-ANXA2 or si-CD147. All of these findings show that the
shedding of microvesicles from tumor cells acts as an efficient
vehicle for CD147 trafficking and that ANXA2 regulates the
transportation of CD147-harboring microvesicle, thereby
contributing to the progression of HCC, although there may be other
molecules in the microvesicles besides CD147.
In conclusion, we report that ANXA2 promotes the migration
and invasion of HCC cells co-cultured with fibroblasts in vitro by
regulating the shedding of CD147-harboring microvesicles from
tumor cells, which contributes to tumor-stroma crosstalk and sheds
light on the mechanisms of ANXA2 in tumor progression.
ANXA2 may be a potential target for the development of effective
therapeutic strategies for the treatment of HCC.
Conceived and designed the experiments: KFD ZNC WPZ. Performed the
experiments: WZ PZ XLX LC DYC. Analyzed the data: KFD WZ PZ
ZNC WPZ. Contributed reagents/materials/analysis tools: XLX ZSS
KST. Wrote the paper: KFD WZ PZ.
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