Silencing of heparanase by siRNA inhibits tumor metastasis and angiogenesis of human breast cancer in vitro and in vivo.

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e1 Cancer Biology & Therapy 2007; Vol. 6 Issue 4
Research Paper
Silencing of Heparanase by siRNA Inhibits Tumor Metastasis
and Angiogenesis of Human Breast Cancer In Vitro and In Vivo
Zhong-Hua Zhang1,3
Yi Chen2
Hua-Jun Zhao2,3
Cheng-Ying Xie2,3
Jian Ding2
Yong-Tai Hou1,*
1Division of Molecular Pharmacology; 2Division of Anti-tumor Pharmacology;
Shanghai Institute of Materia Medica; Shanghai Institutes for Biological Sciences;
3Graduate Schools of the Chinese Academy of Sciences Chinese Academy of
Sciences, Shanghai, P.R. China
*Correspondence to: Yong-Tai Hou; Division of Molecular Pharmacology;
Shanghai Institute of Materia Medica; Shanghai Institutes for Biological Sciences;
Chinese Academy of Sciences; 555 Zu Chong Zi Road; Zhangjiang Hi-Tech Park;
Shanghai 201203 P.R. China; Tel.: +86.21.6203.5047; Fax: +86.21.6203.4655;
Email: ythou@yahoo.com
Original manuscript submitted: 01/09/07
Manuscript accepted: 01/19/07
This manuscript has been published online, prior to printing for Cancer Biology &
Therapy, Volume 6, Issue 4. Definitive page numbers have not been assigned. The
current citation is: Cancer Biol Ther 2007; 6(4):
http://www.landesbioscience.com/journals/cc/abstract.php?id = 3888
Once the issue is complete and page numbers have been assigned, the citation
will change accordingly.
KeY worDs
RNA interference, siRNA, human heparanase,
tumor metastasis, angiogenesis, human breast
cancer, gene silence
ACKnowleDgemenTs
We thank Ms. Li-Juan Lu and Mr. Yong Xi in
the animal center of SIMM for animal experi-
ments.
noTe
Supplemental information can be found
at: www.landesbioscience.com/supplement/
zhangCC6-4-sup.pdf
AbsTrACT
Expression of the heparanase gene is associated with invasive, angiogenic and
metastatic potential of diverse malignant tumors and cell lines. Here we used RNA
interference strategies to evaluate the role of human heparanase in breast malignancy
and to explore the therapeutic potential of its specific targeting. The siRNA targeting
human heparanase almost completely inhibited the expression of heparanase in human
breast carcinoma MDA‑MB‑435 cells, whereas the mismatched siRNA showed no effect.
Cells transfected with heparanase siRNA expressed significantly less heparanase and
profoundly reduced invasion and adhesion in vitro. In MDA‑MB‑435 cell xenograft model,
tumors treated with siRNA were less vascularized and less metastatic than those treated
with saline and the mismatched controls. The association of reduced levels of heparanase
and altered tumorigenic properties in cells with anti‑heparanase siRNA indicates that
heparanase is important in cancer progress and has potential use as a target for
anticancer drug development.
InTroDuCTIon
There is compelling evidence indicating that uncontrolled angiogenesis and metastasis
are major contributing factors in tumor-caused human death.1-3 Heparanase, a mammalian
endo-b-D-glucuronidase, capable of partially depolymerizing heparan sulfate (HS) chains
at a limited number of sites, is proposed to have a promoting role in cancer invasion,
angiogenesis and metastasis.4 It is thought to participate in the cleavage of HS chains from
heparan sulfate proteoglycans (HSPGs) leading to remodeling of the extracellular matrix
(ECM) and to facilitate cell invasion associated with cancer metastasis.5,6 Apart from
its involvement in the egress of cells from the vasculature, heparanase is tightly involved
in angiogenesis, primarily by means of releasing heparin-binding angiogenic factors
sequestered by HS in basement membrane (BM) and ECM, such as bFGF, VEGF, KGF
and HGF, etc.7,8 These promise heparanase as a potential target for inhibiting tumor
metastasis and angiogenesis. RNAi technology, especially chemically synthetic siRNA, is
currently being evaluated not only as an extremely powerful instrument for functional
genomic analyses but also as a potentially useful method to develop highly specific
gene-silencing therapeutics,9,10 whereas limited studies on the application of siRNA to the
organism level are performed.10-15 Recently, Edovitsky et al. reported the use of shRNA
targeting mouse heparanse to reduce the metastatic and angiogenic potential of B16-BL6
mouse melanoma cells in vitro and in vivo.16 However, there are no applications of siRNAs
on human heparanase. In this study, we report the attempt to suppress tumor metastasis
and angiogenesis by human heparanase siRNA in the human breast carcinoma cell line,
MDA-MB-435, and to evaluate its therapeutic significance in a xenograft athymic nude
mouse model.
mATerIAls AnD meTHoDs
Preparation of siRNAs. For effective siRNA fragments screening, five siRNAs (Fig. 1A)
targeting human heparanase (NM_006665) and a GAPDH siRNA (NM_002046) were
prepared by in vitro transcription using SilencerTM siRNA Construction kit (Ambion)
according to the manufacture’s protocol. Oligonucleotides used as the templates of siRNAs
were listed in Table 1S in Supplementary Materials. Transcribed siRNAs were reconstituted
with RNase-Free water to prepare 20 mM stock solutions.
For applications on breast carcinoma MDA-MB-435 cells and animal model of tumor
metastasis, following sequences of chemically modified (U- and C-2'-F) siH1324 and
misH1324 (mismatched siRNA) were synthesized by GenePharma (Shanghai, China):
[Cancer Biology & Therapy 6:4, e1-e10, EPUB Ahead of Print: http://www.landesbioscience.com/journals/cbt/abstract.php?id = 3888; April 2007]; ©2007 Landes Bioscience

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Small Interfering RNA to Human Heparanase
siH1324, 5'-ACUUCGAUCCUUUACCUGA
dTdT-3' (sense), 5'-UCAGGUAAAGGAUCG
AAGUdTdT-3' (antisense), according to 1324-
1344 nt of the sequence of human heparanase
mRNA; misH1324, 5'-ACUUUGAGCCUUU
ACCUGAdTdT-3' (sense), 5'-UCAGGUAAAG
GCUCAAAGUdTdT-3' (antisense), according
to 1254-1274 nt of the sequence of mouse
heparanase mRNA (NM_152803.3).
Transfection of siRNAs. Cells were inoculated
at a density of 1 x 105 cells/well in 24-well plates.
After 24 h, the cells at 70–80% confluence were
transfected with siRNAs (100 nM in final
concentration) and pcHep plasmid (heparanase-
expressing vector derived from pcDNA3.1/
MycHis C (+), well) or siRNAs only (as indicated
concentration) in serum-free medium using
Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
according to the manufacture’s instruction.
Silencing were examined at indicated times
after transfection. Control cells were treated with
the same amount of Lipofectamine 2000 (Mock).
Human heparanase activity assay. siRNA
transfected MDA-MB-435 cells were suspended
in 0.01 M phosphate-citrate buffer (pH 5.8)
and sonicated on ice for 3 x 10 sec. The lysates
were pelleted at 12000 g for 10 min and super-
natants were collected in new Eppendorf tubes.
Protein concentration was determined using the
BCA method. Human heparanase activity assay
was determined toward FITC-HS as previously
described.17,18 For more details, see Supplementary
Materials.
Immunohistochemistry. MDA-MB-435 cells
were seeded on glass coverslips (18 mm x 18 mm)
coated with 0.1 mg/ml poly-L-lysine solution
in 6-well plates and transfected with siH1324
(50 and 200 nM) or misH1324 (200 nM). After
36 h, the cells were washed twice with PBS
and fixed with prechilled 4% paraformaldehyde
for 10 min. The cells were then washed twice
with cold PBS and the intrinsic fluorescence
was blocked by treating the cells with 50 mM
NH4Cl for five min at room temperature, and
then washed twice with PBS. Slides were
incubated overnight at 4°C with polyclonal
anti-heparanase antibody (1:5000, from rabbit
immunized with recombinant heparanase
expressed in Escherichia coli), washed three times
with PBS, and incubated with FITC-conjugated
goat anti-rabbit immunoglobulin G (1:100
diluted in PBS, Santa Cruz Biotechnologies Inc., CA) for 45 min
at 24°C. After washed three times with PBS, slides were mounted
with 50% glycerol and 1 mg/ml p-phenylendiamine in 1 x PBS, and
examined microscopically with an Olympus fluorescent microscope.
Tissue sections (5 mm thick) for immunostaining were obtained
from formalin-fixed and paraffin-embedded primary tumors
produced in mice by injecting MDA-MB-435 cells into mammary
fat pads (m.f.p.) and treated with siH1324 and misH1324 siRNAs.
The sections were treated with xylene and rehydrated, endogenous
peroxidase activity was quenched by incubation with 3% H2O2 for
10 min. After washed in distilled water, the sections were incubated with
5% BSA for 30 min, and incubated with rabbit polyclonal anti-CD31
antibody (Santa Cruz Biotechnologies, Inc.) diluted 1:100 at 4°C
overnight. After washed twice with PBS, slides were incubated with
biotin-labeled rabbit IgG (Santa Cruz Biotechnologies, Inc.) for
30 minutes followed with streptavidin-HRP for 20 min at room
temperature. Lastly, sections were developed with three, 30-diami-
no-benzidine and stained with hematoxylin.
Figure 1. Reduction of recombinant and endogenous heparanase protein in human cell lines by
siRNAs. (A) Schematic depiction of five heparanase siRNAs. (B) Left panels: Western blot analysis.
siRNAs and pcHep plasmid were cotransfected in HEK 293 cells with Lipofectamine 2000
or without siRNAs (Mock) for 36 h. The same membrane was stained with antibodies against
Myc‑Tag and b‑actin. The latter served as an equal loading control. Right panels: Quantification
of Western blots normalized to the level of b‑actin. (C) Dose kinetics: HEK 293 cells were
transfected with pcHep plasmid and indicated concentration of siH1324 and misH1324, for
36 h. Cells were harvested for Western blot analysis. (D) Validation of the effect of siH1324
and misH1324 on endogenous heparanase in human breast carcinoma MDA‑MB‑435 cells.
(E) Schematic depiction of the sequences of chemically synthesized siH1324 and misH1324.
(F) Time kinetics: Left panels: HEK 293 cells were transfected with pcHep plasmid and 200 nM
siH1324 for indicated time points and analyzed by Western blot with Myc‑Tag and b‑actin anti‑
bodies. Right panels: MDA‑MB‑435 cells were transfected with 200 nM siH1324 for indicated
time points and stained with self‑prepared heparanase and b‑actin antibodies.

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e3 Cancer Biology & Therapy 2007; Vol. 6 Issue 4
Cell proliferation assay. MDA-MB-435 cells were transfected in
100 mm dishes with siH1324 and misH1324 siRNAs. 24 h post-
transfection, the cells were trypsinized and diluted to 5 x 105 cells/ml.
The cells were then seeded in complete media in triplicate in 60-mm
dishes at the density of 5 x 105 cells per dish. The cells were dissociated
into single cell suspension with trypsin and EDTA solution and
counted in triplicate with a Coulter counter (Coulter Electronics,
Luton, UK) every day for four days.
Cell adhesion assay. siRNA-transfected MDA-MB-435 cells were
suspended in complete medium and inoculated into ECM-coated
wells (2 × 104 cells per well in 96-well plates precoated with 100 ml
of 20 mg/ml Matrigel) in triplicate, and incubated at 37°C in
serum-free complete medium (pH 7.2) for 2 h. After incubation, the
wells were washed three times with PBS and the remaining cells were
fixed in 4% paraformaldehyde for 20 min at room temperature. The
cells were stained with 0.1% crystal violet and washed three times
with PBS to remove free dye. After extraction with 10% acetic acid,
absorbance of the samples were measured at 570 nm. 0%, 20%,
50% and 100% of inoculated cells were directly fixed in 4%
paraformaldehyde 2 h after inoculation.
Cell wound healing assay. MDA-MB-435 cells were transfected
in 24-well plate with siH1324 and misH1324 siRNAs. Twenty-four
hours posttransfection, the cells were scraped with the fine end of
1-ml pipette tips (time 0). Plates were washed twice with PBS to
remove detached cells and incubated with the complete growth
medium, and cell migration into the wounded empty space was
followed after 24 h.19
Matrigel invasion assay. Invasion assay was performed using
modified Boyden chambers with a polycarbonate nucleopore
membrane (Corning Inc., Corning, NY). Filters (6.5-mm in diameter,
8-mm pore size) were coated with 100 ml of 1 mg/ml Matrigel
(dissolved in serum-free DMEM medium; Becton-Dickinson
Sciences). 600 ml of DMEM medium containing 10% FBS was added
to the lower chambers. siRNA-transfected cells (1 x 105 cells/well)
were added to the upper chambers and allowed to invade for 24 h at
37°C in a CO2 incubator. Unmigrated cells were removed from the
upper chamber with a cotton swab. The remaining cells were fixed,
stained with 0.1% crystal violet for 10 min at room temperature, and
photographed under microscope and measured at 570 nm after
extraction with 10% acetic acid for 10 min.20
For HUVECs migration assay, filters in the upper chamber were
coated with 1% gelatin solution. The lower chamber were filled
with 600 ml of M199 medium containing 1% FBS, which was
preconditioned 48 h with siRNA-transfected MDA-MB-435 cells
growing in 10-cm dishes coated with 3 ml of 10 mg/ml Matrigel,
filtered with 0.22 mm membrane, and then examined for bFGF
levels with an Quantikine® bFGF Immunoassay kit (R&D Systems,
Minneapolis, MN). HUVECs (5 × 104 cells/well) were seeded in the
inner chamber in M199 containing 1% FBS. After incubation for
24 h at 37°C, unmigrated cells were removed. The migrated cells
were fixed and stained with 0.1% crystal violet, then extracted with
10% acetic acid for 10 min and measured at 570 nm.18
In vitro HUVEC tube formation assay. Matrigel was distributed
in a 96-well plate (50 ml/well) and allowed to solidify for 2 h at
37°C. HUVECs (passes 2–6) were serum starved in M199 medium
for 2 h. The cells were suspended in M199 medium preconditioned
with siRNA-transfected MDA-MB-435 cells and added to the
Matrigel-coated wells at the density of 5 x 104 cells/well. The cultures
were incubated at 37°C for 8 h. Tube formation was observed, and
digital pictures were captured. Quantification of antiangiogenic
activity was calculated by measuring the length of tube walls formed
between discrete endothelial cells in each well relative to the control
(Mock transfection). Total length in three high-powered fields was
measured per well in a blind manner.
Gene microarray. Analyses were performed on the Human
14K Expression cDNA Array (V2.0) chip (Shanghai BioChip Inc.,
Shanghai, China). Transfected cells were used for RNA extraction
and RT preparation of fluorescent cDNA probes labeled with Cy3-
(200 nM siH1324) or Cy5- (200 nM misH1324) dCTP (Amersham
Pharmacia Biotech), respectively. Quantified probes were applied
onto the prehybridized chips under a cover glass. After hybridization
and washing, the chips were scanned with an Aglient Array Scanner
G2655AA (Agilent Technologies Inc., Santa Clara, CA) at two
wavelength (532 nm for Cy3 and 653 nm for Cy5). The resulting
images were analyzed using Imagene software (BioDiscovery,
Los Angeles, CA). Overall intensities were normalized using the
Genespring software (Silicon Genetics, Redwood, CA). We applied a
cutoff intensity ratio of siH1324:misH1324 at 2 and 0.5 (p < 0.05,
student’s t-test) for up and downregulated genes, respectively.
Experiments were performed three times, and data sets were main-
tained in a Microsoft Excel spreadsheet.
In vivo spontaneous metastasis assay. Female athymic nude
mice (BALB/cA nu/nu) aged 4–5 weeks were obtained from
Shanghai Institute of Materia Medica, and housed in sterile cages
under laminar airflow hoods in a specific pathogen-free room
with a 12 h light and 12 h dark schedule, and fed autoclaved
chow and water ad libitum. All experiments were performed
according to the institute’s ethical guidelines on animal care.
MDA-MB-435 cells were orthopedically injected into the
mammary fat pads (m.f.p.) of the nude mice aged 4–5 weeks.
Mice were anesthetized with chloral hydrate, and 5 mm incisions
were made in skin over the lateral thorax, as described previ-
ously.21,22 The m.f.p.s were exposed, and inoculums of 5 x 106
cells/0.1 ml were implanted into the tissue through a 27-gauge
needle. Skin incisions were closed with wound chips and removed
one week later. At a volume of 100-200 mm3, mice were divided into
four experimental groups after balancing tumor volumes, specifically:
(a) untreated (n = 6, i.v. 100 ml of saline per mouse); (b) ADM (n = 6,
i.v. 5 mg/kg Adriamycin, used for a positive control); (c) 10 mM
siH1324 (n = 6, in primary tumor, 100 ml of the mixture of 20 mg
siH1324 and 40 mg Lipofectaime 2000 in Opti-MEM I per mouse,
about 1 mg/kg); (d) 10 mM misH1324 (n = 6, in primary tumor,
100 ml of the mixture of 20 mg misH1324 and 40 mg Lipofectaime
2000 in Opti-MEM I per mouse, about 1 mg/kg). The samples were
administrated every three days for seven weeks thereafter. Tumor
size was measured before each injection with micro calipers. Tumor
volume was calculated according to the formula: length x width
x width x 0.5, and presented as RTV (Relative Tumor Volume)
= Tumor Volume (day after initial treatment)/ Tumor Volume (day of
initial treatment). Mice were sacrificed by cervical dislocation three
days after the final therapy. The lungs were removed and fixed with
Bouin’s solution for 24 h, and metastasis nodules on lungs were
counted under a dissecting microscope. The primary tumors were
extirpated and cut into two parts. One part of the tumors was chilled
in liquid nitrogen and stored at -80°C, and total RNAs were
extracted from these samples and analyzed by RT-PCR. The other
part was fixed in 10% neutral-buffered formalin solution, paraffin
embedded, sectioned and immuno-stained with anti-CD31
polyclonal antibody for vascularization detection.
Statistic. Results are represented as mean values ± SD or ± SE. The
significance of differences between means was assessed by Student’s
t-test. P-values of <0.05 were regarded as statistically significant.

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Small Interfering RNA to Human Heparanase
Supporting material and methods. For details
about cell culture, Western blotting assay, reverse
transcription PCR and quantitative RT-PCR, human
heparanase activity assay, see Supplementary Materials.
resulTs
Effect of heparanse siRNAs on the exprssion of
heparanase in human cell lines. siRNA can silence gene
expression in a sequence-specific manner. Since different
siRNAs targeting the same gene vary in efficiency,9
we designed five siRNAs against the open reading
frame of human heparanase mRNA, as illustrated in
Figure 1A. The five siRNAs were tested to inhibit the
expression of cotransfected recombinant heparanase
in HEK 293 cells and endogenous heparanase in
breast cancer cells MDA-MB-435.
To examine the effect of the five siRNAs on hepa-
ranase expression in human cell lines, Western blot
analysis was performed on the cell lines 36 h after
transfection with siRNAs. Since no commercial
anti-heparanase antibody was available at the time
of the experiment, we first screened effective siRNAs
by cotransfecting the heparanase expressing plasmid
and siRNAs in HEK 293 cells and detecting the
expression of recombinant heparanase (fused with
C-terminal Myc-Tag) with Myc-Tag antibody
(Fig. 1B). The protein level of heparanase was
standardized to the levels of b-actin. Treatment with the
control siGAPDH or siH1637 had almost no effect.
siH492, siH879 and siH887 appeared to be effective in
inhibiting heparanase expression, whereas siH1324 was
highly effective (96.8% inhibition).
For further application, U, C-2’-F modified siH1324 and
mismatched misH1324 were chemically synthesized. The inhibition
effects of siH1324 and misH1324 on heparanase expression
were tested in HEK 293 and MDA-MB-435 cells. Cotransfected
with pcHep plasmid, 1 nM siH1324 reduced the level of hepa-
ranase protein to less than 10% compared to the control. The
inhibition was positively correlated with the amount of siH1324.
Only 1–5% of heparanase protein remained after incubation with
50 nM siH1324. After treatment with 100 nM and 200 nM siH1324,
the expression of heparanase was undetectable by Western blot even
with long time exposure (Fig. 1C). Therefore, 50 nM and 200 nM
siH1324 were used in further experiments. As the mismatched
negative control, misH1324 in different concentrations had no effect
on the expression of recombinant heparanase (Fig. 1C and D),
indicating that 2-nt mutation could totally block the effect of
siH1324 in the experiment (Fig. 1E). Similar results were obtained
in breast carcinoma MDA-MB-435 cells, which have high-level
expression of endogenous heparanase (Fig. 1D).23 misH1324 also
showed no effect on the mouse heparanase in mouse melanoma
B16-L6 cells (data not shown).
We investigated how long the effect of siH1324 would maintain
in human cells. After transfection with 200 nM of siH1324, the
expression of heparanase was severely inhibited in HEK 293 and
MDA-MB-435 cells. The expression of heparanase reached the
bottom 48 h post-transfection, and then began to recover. However,
the inhibition effect of siH1324 maintained more than 80% even
96 h post-transfection in both cell lines (Fig. 1F). It indicated that
siH1324 could silence the expression of heparanase for about four
days and allowed enough time for further detection of the phenotype
variation caused by inhibiting the expression of heparanase in
MDA-MB-435 cells.
Heparanase siH1324 reduces the amount and activity of
heparanase in MDA‑MB‑435 cells. Immuno-fluorescent assay was
used to detect the expression of heparanase in MDA-MB-435 cells.
Heparanase had strong, similar expression profiles in normal, mock-
and 200 nM misH1324-transfected samples. Fifty nM siH1324
treatment greatly decreased the intensity of fluorescence and 200 nM
siH1324 almost quenched the fluorescence to background (Fig. 2A).
Using a size exclusion chromatography assay along with a FITC-HS,
we examined the ability of heparanase siRNA to inhibit hepa-
ranase-mediated degradation of heparanase sulfate. The lysates of
MDA-MB-435 cells transfected with siH1324 were incubated with
FITC-HS and loaded on HPLC. The resulting chromatographic
profiles indicated that siH1324 significantly and dose-dependently
inhibited heparanase enzymatic activity (Fig. 2B). Fifty nM siH1324
reduced heparanase activity significantly, and 200 nM totally
blocked the activity of heparanase. 200 nM misH1324 transfected
samples had the elution profile identical to that of mock samples
(Data not shown). Comparing with the results of the Western blot,
50 nM siH1324 transfected MDA-MB-435 cells still showed some
activity in degrading HS-FITC, which might be caused by variations
of the transfection efficiency and long time incubation with the
substrate. These results indicated that siH1324 effectively blocked the
expression of heparanase in MDA-MB-435 cell line and misH1324
could act as an eligible negative control.
Figure 2. Reduction of the expression and activity of heparanase in MDA‑MB‑435 cells by
siH1324. (A) Fluorescent immunostaining: MDA‑MB‑435 cells were treated with indicated
siRNAs for 48 h and stained with heparanase antibody, then photographed under a
fluorescent microscope. (B) HPLC chromatography: MDA‑MB‑435 cells were treated with
indicated siRNAs for 48 h and used for heparanase enzymatic activity assay by HPLC
methods as described in supplementary materials.

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Heparanase siH1324 inhibits the proliferation, adhesion,
migration and invasion of MDA‑MB‑435 cells. In our previous
unpublished work, we found that NIH 3T3 and CHO cells
transfected with heparanase expression vector grew faster, and adhered
quicker to Matrigel than untransfected cells. Therefore, we tested the
effect of heparanase siRNA on the proliferation of MDA-MB-435
cells. As illustrated in Figure 3A, cells transfected with 50 nM
siH1324 (6.13 ± 0.32 x 106 cells at 96 h) had a similar growth
rate with 200 nM misH1324 treated groups (6.15 ± 0.49 x 106
cells at 96 h). After treatment with 200 nM siH1324, the prolif-
eration of MDA-MB-435 cells was inhibited 33.2% compared with
the mock transfection groups (4.88 ± 0.11 x 106 cells versus 7.30 ±
0.28 × 106 cells at 96 h, p < 0.05). Further, in the adhesion assay,
cells treated with 50 nM and 200 nM siH1324 exhibited markedly
reduced ability in adhesion to the precoated Matrigel, 67.5%
and 57.5% compared to mock group, respectively. However, the
200 nM misH1324 treated cells had similar ability in adhesion as
mock (Fig. 3B). These suggested heparanase siRNA suppressed
heparanase-associated cell proliferation and adhesion in vitro.
As heparanase expression has been consistently correlated with the
metastasis potential of tumor cells,16,23 we used cell wound healing
assay to examine the effect of siRNAs on heparanase-associated cell
migration in MDA-MB-435 cells. As shown in Figure 3C, cells in
the mock and 200 nM misH1324 treated groups almost healed
the wound 24 h after scraping with pipette tips. Cells exposed
to 50 nM and 200 nM siH1324 showed reduced rate of wound
healing, suggesting that siH1324 inhibited heparanase-associated
cell migration. To confirm and extend the above findings, we exam-
ined whether siH1324 abrogated the invasiveness of MDA-MB-435
cells. In the absence of siH1324, the MDA-MB-435 cells freely
invaded the Matrigel and passed into the lower chamber. Treatment
of the cells with 50 nM and 200 nM siH1324 significantly and dose-
dependently reduced the number of migrated cells, with 200 nM
siH1324 yielding 49.6% inhibition compared with mock transfected
cells (Fig. 3D). Collectively, these findings indicated that siH1324
inhibited heparanase-associated metastasis in vitro.
Heparanase siH1324 attenuates heparanase‑related angiogenesis
in vitro. Heparanase has been identified as one of potential positive
regulators of angiogenesis.7,8 Inhibition of heparanase activity has
been shown to inhibit both tumor growth and metastasis in a variety
of animal tumor models.24-27 Hereby, we exposed HUVEC cells to
medium conditioned with siRNA-treated MDA-MB-435 cells and
investigated their ability of migration and tube formation in vitro.
Results of transwell chamber assays shown in Figure 4A revealed
that the migration of HUVEC cells in 200 nM siH1324 group
was inhibited 18.7% and 17.2% comparing with that of mock and
200 nM misH1324 treated groups, respectively. In the tubular
network formation test, the tubes formed in the test groups had
similar shapes, whereas the total length of the vascular cord in the
200 nM siH1324 groups was significantly shorter than other groups
(Fig. 4B). We further investigated the reason for the former results
by detecting the concentration of bFGF in the conditioned medium
(Fig. 4C). Nearly 0.5 ng bFGF was released from Matrigel coating
the surface area of a 6-cm dish by mock and 200 nM misH1324
transfected MDA-MB-435 cells. Treatment with 200 nM siH1324
reduced the amount of bFGF to 0.37 ± 0.09 ng, suggesting that
siH1324 might suppress the heparanase-associated angiogenesis in
vitro by regulating the amount of bFGF from ECM.
Effect of heparanase siH1324 on the gene spectrum of
MDA‑MB‑435 cells. To identify genes associated with altered
tumorigenic properties of MDA-MB-435 cells initiated by silencing
heparanase, we compared gene expression profiles between MDA-
MB-435 cells treated with 200 nM siH1324 and 200 nM misH1324
by means of microarray. We found that heparanase siH1324 induced
diverse expression of many genes relating to cell proliferation,
shock response, metabolism and gene transcription and transla-
tion, even other metastasis-related genes (Table 1 and Table 3S
in Supplementary Materials). Results from quantitative RT-PCR
tests validated the change of gene expression spectrum. Among
these genes, proteoglycan 2 (PRG2), homologous with lectin
and important in cell proliferation, was upregulated the most,
while cell division cycle 16 (CDC16, an important component of
anaphase-promoting complex) and heat shock protein 86 (HSP86,
also known as official symbol HSP90AA1, a molecular chaperone
for the conformation maturation of oncogenic signaling proteins)
were mostly downregulated in MDA-MB-435 cells 48 h after treat-
ment with siH1324. Intriguingly, expression of lanosterol synthase
(LSS) and cathepsin D (CTSD), associating with metabolism
and protein processing, were also enhanced after transfection.
No change of bFGF and VEGF gene expressions was observed,
thus heparanase might regulate the progress of angiogenesis
by releasing the angiogenic factors from ECM or the surface
Table 1 Gene expression profiles of MDA-MB-435 cells treated with heparanase siH1324 versus misH1324
(ratio>2.0 and ratio<0.5, p < 0.05)
genbank ID unigene ID symbol Product ratio P-value qPCr
upregulated gene list (+: up; ‑: down)
NM_002728 Hs.99962 PRG2 proteoglycan 2 3.778 0.000123 (+3.5 ± 2.0)
S81221 Hs.93199 LSS lanosterol synthase 2.416 0.000971
NM_002340 Hs.93199 LSS lanosterol synthase (2,3‑oxidosqualene‑lanosterol cyclase) 2.107 0.00195 (+2.5 ± 1.1)
NM_001909 Hs.343475 CTSD Homo sapiens cathepsin D (lysosomal aspartyl protease) 2.078 0.00273 (+3.0 ± 1.5)
NM_182917 Hs.433750 EIF4G1 eukaryotic protein synthesis initiation factor 2.041 0.00102 (+2.2 ± 1.2)
NM_006665 Hs.44227 HPSE Homo sapiens heparanase (HPSE), mRNA (‑57.8 ± 1.4)
Downregulated gene list
AF187554 Hs.279789 GPI sperm antigen‑36,glucose phosphate isomerase 0.241 0.0166 (‑5.5 ± 2.1)
AV743808 Hs.381246 HSP90AA1 Human heat shock protein 86 mRNA, 5’end 0.317 0.0162 (‑4.0 ± 1.5)
NM_152601 Hs.306478 ZNF564 zinc finger protein 564 0.412 0.0199
NM_003903 Hs.1592 CDC16 CDC16 cell division cycle 16 homolog (S. cerevisiae) 0.313 0.0157 (‑2.8 ± 1.5)
CD239654 Hs.479670 ARF6 ADP‑ribosylation factor guanine nucleotide factor 6 0.388 0.0306
AF203815 Hs.355998 MALAT1 alpha gene sequence 0.308 0.0157 (‑3.5 ± 2.0)
BX538238 Hs.187199 PRO1073 PRO1073 protein 0.296 0.0211 (‑3.5 ± 2.1)