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SUPPORTING MATERIAL
MicroRNA-29b Suppresses Tumor Angiogenesis, Invasion and
Metastasis by Regulating MMP-2 Expression
Fang, et al.
Index:
1. Supporting Materials and Methods……………………… Page 2 - 6
2. Supporting Reference……………………………………. Page 7
3. Supporting Figure Legends……………………………… Page 8 - 12
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Supporting Materials and Methods
Establishment of miR-29b stably expressing cell line
LM6 subline with Tet-off-inducible miR-29b expression (LM6-miR-29b) and its
control line (LM6-vec) were established using the Tet-off system (Clontech, Palo Alto,
CA) as follows. LM6 cells were first infected with the retrovirus expressing the
tetracycline (Tet)-controlled transactivator (pRetroX-Tet-Off Advanced), followed by
selection for the LM6-Tet-Off stable line using 500 μg/ml of G418. The LM6-Tet-Off
cells were subsequently infected with the retroviruses that expressed hsa-miR-29b
(pRetroX-miR-29b) or empty vector (pRetroX-Tight Control) under the control of a
Tet-regulated promoter. The infected cells were then subjected to selection with both
G418 (Clontech, 500 μg/ml) and puromycin (Clontech, 0.5 μg/ml) in the presence of
Dox (Clontech, 0.1 μg/ml). The resulting stable lines were maintained in DMEM
supplemented with 10% FBS, 100 μg/ml of G418, 0.25 μg/ml of puromycin and 0.1
μg/ml of Dox.
Isolation of Human umbilical vein endothelial cells (HUVECs)
The vein of each umbilical cord was cannulated and rinsed with 1×PBS three times,
and then perfused and incubated with 0.25% trypsin/0.04% EDTA (AMRESCO, Ohio)
for 15 min at 37°C with the ends of the vein clamped. The trypsin/EDTA solution was
then collected in a sterile tube. To further detach all HUVECs, M-199 (Invitrogen)
supplemented with 200 U/ml penicillin, 200 µg/ml streptomycin and 10% FBS, was
applied to flush the vein thoroughly. The washout was then collected in the same
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sterile tube. After centrifugation at 500g for 5 min, HUVECs were re-suspended in
Serum Free Medium for Endothelial Cells (SFM, Invitrogen), supplemented with 20%
FBS, 0.1 mg/ml of heparin and 0.03 mg/ml of endothelial cell growth supplement
(Upstate Biotechnology; Lake Placid, NY).
RNA oligoribonucleotides and vectors
For the in vivo subcutaneous tumorigenicity assay, all pyrimidine nucleotides in NC
or miR-29b duplex were substituted by their 2’-O-methyl analogs to improve RNA
stability.1 The miRIDIAN microRNA Hairpin Inhibitor of miR-29b (anti-miR-29b)
and miRIDIAN microRNA Hairpin Inhibitor Negative Control #2 (anti-miR-C) were
obtained from Dharmacon (Chicago, IL).
Construction of vectors was performed as follows. The expression vectors for
miR-29b were generated by cloning the genomic fragments (439 bp) encompassing
the corresponding miR-29b precursor and its 5’- and 3’-flanking sequences into
pcDNA3.0 (Invitrogen) or pRetroX-Tight-Pur (Clontech). The resulting vectors were
designated
as
pc3-miR-29b
and
pRetroX-miR-29b,
respectively.
For
pGL3cm-MMP2-3'UTR-WT, a wild-type 3'-UTR segment (1121 bp) of human
MMP-2 mRNA that contained a putative binding site for miR-29b was PCR-amplified
and inserted into the EcoRI and XbaI sites downstream of the stop codon of firefly
luciferase in pGL3cm,2 which was created based upon the pGL3-control (Promega,
Madison, WI). The plasmid, pGL3cm-MMP2-3'UTR-MUT, which carried the
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mutated sequence in the complementary site for the seed region of miR-29b, was
generated by site-specific mutagenesis based on pGL3cm-MMP2-3'UTR-WT.
MMP-2 expression vectors pc3-MMP2 was constructed by cloning the coding
sequence of MMP2 (1983 bp) into EcoRI and XhoI sites of pcDNA3.0. All constructs
were verified by direct sequencing. The sequences of primers used for PCR are
provided in Supporting Table 1.
In vitro assays of migration and invasion
The migration and invasion assays were done in 24-well Boyden chamber with 8 μm
pore size polycarbonate membrane (Corning, NY). For invasion assay, the membrane
was coated with 15 μg Matrigel (R&D) to form a matrix barrier. Thirty-six hours after
transfection with RNA duplex, tumor cells (2105 LM6 or 8104 H2M in 100 μl
serum-free medium) were added to the upper compartment of the chamber, while the
lower compartment was filled with 600 μl of DMEM containing 10% FBS. After
incubation at 37°C for 24h, tumor cells remaining on the upper surface of the
membrane were removed. The cells on the lower surface of the membrane were fixed,
stained with crystal violet and then counted under a light microscope. For migration
assay, only one third of cells were applied to the transwell chamber without Matrigel.
Wound healing assay was applied to examine the migration of HUVECs. HUVECs
grown to monolayer were scratched by a pipette tip, and then rinsed, followed by
culture in the presence of 75% TCM for 12h. Migration distance (in μm) was
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determined as the reduction of the width of the open area.
Analysis of Cell Proliferation
BrdU incorporation assay was employed to assess the proliferation of HUVECs after
incubation with TCM. HUVECs were grown in SFM for 12h, then cultured in the
presence of 75% TCM containing 40 μM BrdU for 1h, washed with 1×PBS thrice,
fixed in 70% ethanol supplemented with 20 mM glycine (pH 2.0) at room temperature
for 45 min, followed by incubation sequentially with mouse mAb against BrdU
(BrdUAb-3, Neomarkers, Fremont, CA) and goat-anti-mouse Alexa-488-conjugated
secondary antibody (Invitrogen), each for 1 h at 37°C. Three washes in 1×PBS were
performed, after each incubation with antibody. The cells with BrdU incorporation
was observed under a fluorescent microscope. At least 500 cells were counted for
each sample.
Detection of MMP-2 activity in TCM by gelatin zymography
To analyze the gelatinolytic activity of MMP-2, aliquots of TCM were mixed with 4×
nonreducing sample buffer (250 mM Tris, 2.5% SDS, 12.5% glycerol, 0.1%
bromophenol blue) and directly applied to a 10% acrylamide gel containing 1 mg/ml
gelatin. After electrophoresis, the gel was soaked in 2.5% Triton X-100 to remove
SDS and then incubated at 37°C overnight in the development buffer (50 mM
Tris-HCl, pH 7.4, 0.2 M NaCl, 5 mM CaCl2, 0.02% NaN3), followed by staining
with Coomassie Brilliant Blue R-250.
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Immunohistochemical staining (IHC) for MMP-2 and CD34
OCT-embedded Matrigel plugs and paraffin-embedded tissues were cut into 5 μm
sections
and
placed
on
polylysine-coated
slides.
Paraffin
sections
were
de-paraffinized in xylene, rehydrated through graded ethanol, quenched for
endogenous peroxidase activity in 0.3% hydrogen peroxide, and processed for antigen
retrieval by microwave heating in 10 mM citrate buffer (pH 6.0). Sections were
incubated at 4°C overnight with mouse mAb against human MMP-2 (cat. 35-1300Z,
Invitrogen) or CD34 (cat. sc-52312, Santa Cruz Biotechnology, Santa Cruz, CA) or
rat mAb for mouse CD34 (cat. 119301, BioLegend, San Diego, CA). Immunostaining
was
performed
using
the
ChemMate
DAKO
EnVision
Detection
Kit,
peroxidase/DAB/rabbit/mouse (code K 5007, DakoCytomation, Glostrup, Denmark)
for human MMP-2 and CD34, or donkey anti-rat IgG-HRP (cat. 6430,
Southernbiotech, Birmingham, AL) for mouse CD34, which resulted in a
brown-colored precipitate at the antigen site. Subsequently, sections were
counterstained with hematoxylin (Zymed Laboratories, South San Francisco, CA) and
mounted in a non-aqueous mounting medium. All runs included a no primary
antibody control.
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Supporting References:
1.
Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C, Langkjaer N, et al. A large-scale
chemical modification screen identifies design rules to generate siRNAs with high activity,
high stability and low toxicity. Nucleic Acids Res 2009;37:2867-2881.
2.
Su H, Yang JR, Xu T, Huang J, Xu L, Yuan Y, et al. MicroRNA-101, down-regulated in
hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity. Cancer Res
2009;69:1135-1142.
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Supporting Figure Legends
Supporting Fig. 1. Restoration of miR-29b inhibits tumor cell-promoted tube
formation of HUVECs. HUVECs were cultured in the presence of 75% TCM from
95D (A) or HCT116 (B) cells without transfection (panel 1) or transfected with NC
(panel 2) or miR-29b (panel 3). Representative images of tube formation and the
number of branch points of HUVECs are presented. Scale bar, 50 µm. *, P <0.05.
Supporting Fig. 2. Restoration of miR-29b inhibits LM6 cell-promoted HUVECs
proliferation. HUVECs were serum deprived for 12h, then incubated for 1h with 40
μM BrdU in the presence of 75% TCM that were derived from LM6 cells without
transfection or transfected with NC- or miR-29b duplex. The y axis indicates the
percentage of BrdU positive cells. *, P <0.05.
Supporting Fig. 3. Restoration of miR-29b inhibits LM6 cell-promoted HUVECs
migration. HUVECs grown to monolayer were scratched and then cultured in the
presence of 75% indicated TCM derived from LM6 cells for 12h. Images were
acquired directly after scratching (0h) and 12h later. Migration distance was
determined as the reduction of the width of the open area between 0h and 12h. Dotted
lines indicate the front of migration. Scale bar = 100 μm. **, P <0.01.
Supporting Fig. 4. Antagonism of miR-29b decreases the endogenous level of
miR-29b. Forty-eight hours after transfection, LM6 cells transfected with anti-miR-C
or anti-miR-29b were applied to qPCR assay. The miR-29b level was normalized to
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RNU6B expression. No error bar is shown for anti-miR-C-transfectants, because its
normalized miR-29b level was set to 1. **, P <0.01.
Supporting Fig. 5. Antagonism of miR-29b enhanced the pro-angiogenic activity and
invasive capacity of HCC cells. (A) Analysis of angiogenesis by capillary tube
formation assay. HUVECs were cultured in the presence of 75% TCM from LM6
cells transfected with anti-miR-C (panel 1) or anti-miR-29b (panel 2). Representative
images of tube formation and the number of branch points of HUVECs are presented.
(B) Analysis of tumor invasion by transwell chamber. LM6 cells transfected with
anti-miR-C (panel 1) or anti-miR-29b (panel 2) were applied to transwell chamber
coated with Matrigel and then incubated for 24h. Scale bar, 100µm. *, P <0.05.
Supporting Fig. 6. miR-29b suppresses tumor growth in subcutaneous xenograft
model. LM6 cells that were transiently transfected with NC or miR-29b duplex were
injected subcutaneously into either side of the posterior flank of the same nude mice.
Photographs of dissected tumors (A) and the curves of tumor growth (B) are shown.
Tumor volume (V) was monitored over a course of 35 days, by measuring the length
(L) and width (W) of the tumor with calipers and calculated using the formula: V =
(LW2) 0.5. Wilcoxon Signed-Ranks Test was used to compare tumor volumes. *, P
<0.05.
Supporting Fig. 7. Analysis of miR-29b expression in LM6-miR-29b and LM6-vec
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cell lines by qPCR. LM6-miR-29b were cultured with (+) or without (-) Dox for 48h.
The miR-29b level was normalized to RNU6B expression. No error bar is shown for
LM6-vec (control), because its normalized miR-29b level was set to 1. *, P <0.05.
Supporting Fig. 8. miR-29b suppresses tumor growth in orthotopic xenograft model.
Tumors were developed in the implanted sites in 11/14 mice in LM6-vec (control)
group, 9/14 and 7/9 mice in miR-29b-early and -late induction groups, respectively.
Tumor was measured for its length (L) and width (W) with calipers. Tumor volume
(V) was calculated using the formula: V = (LW2) 0.5. The volume for the case
without tumor development was counted as zero. *, P <0.05.
Supporting Fig. 9. Analysis of MMP-2 activity in TCM by gelatin zymography.
TCM was collected from LM6 cells transfected with NC/vector (lane 1),
miR-29b/vector (lane 2), NC/MMP2 (lane 3) or miR-29b/MMP2 (lane 4). The results
were reproducible in three independent experiments and the representative images are
shown.
Supporting Fig. 10. miR-29b expression is reversely correlated with MMP2 level
and MVD in 127 human HCC tissues. Level of MMP2 analyzed by
immunohistochemical staining was quantified using modified Histo-score. MVD was
evaluated based on mCD34 staining. The mature miR-29b level was examined by
qPCR and normalized to U6 expression. Statistical analysis was performed using
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Pearson’s correlation coefficient.
Supporting Fig. 11. Knockdown of TIMP-2 abrogated the anti-angiogenic effect of
miR-29b. (A) Knockdown of TIMP-2 by si-TIMP2. LM6 cells without transfection
(lane 1) or transfected with NC (lane 2) or si-TIMP2 (lane 3) were applied to RT-PCR
analysis for the expression of TIMP-2. β-actin was amplified in the same reaction as
internal control. (B) Knockdown of TIMP-2 abrogated the anti-angiogenesis effect of
miR-29b. HUVECs were cultured in the presence of 75% TCM from LM6 cells
co-transfected with the indicated RNA duplexes. Representative images of tube
formation and the number of branch points of HUVECs are presented. Scale bar, 100
µm. **, P <0.01.
Supporting Fig. 12. Knockdown of MMP-2 by si-MMP2. (A) Cellular level of
MMP-2 was dramatically reduced by si-MMP2. HCC cells without transfection (lane
1) or transfected with NC (lane 2) or si-MMP2 (lane 3) were applied to RT-PCR
analysis for the expression of MMP-2. β-actin was amplified in the same reaction as
internal control. (B) MMP-2 activity in TCM was diminished by si-MMP2. TCM was
collected from HCC cells without transfection (lane 1) or from cells transfected with
NC (lane 2) or with si-MMP2 (lane 3).
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Supporting Fig. 13. Blocking MMP-2 in TCM mimicked the suppressive effect of
miR-29b on VEGFR2-signaling. HUVECs were cultured in the presence of TCM
from LM6 cells without treatment (lane 1) or pre-incubated with control IgG (lane 2)
or MMP-2-neutralizing antibody (lane 3). HUVECs were grown in SFM
supplemented with 1% FBS for 12h and then cultured in the presence of SFM or 75%
TCM for 20 min, before immunoblotting analysis for phosphor-Tyr1175-VEGFR2,
VEGFR2, phospho-ser473-AKT, AKT, phosphor-T202/Y204-ERK1/2 and ERK1/2.
All results were reproducible in three independent experiments and the representative
immunoblots are shown. β-actin, internal control.
Supporting Fig. 14. miR-29b restoration displays no effect on VEGFA level of TCM.
SFM, and TCM that was collected from HCC cells without transfection or from cells
transfected with NC, miR-29b or si-MMP2, were applied to ELISA assay for VEGFA.
The optical density (OD) represents the level of VEGFA. SFM was used as blank
control, and the OD of SFM was subtracted from the OD of TCM. No error bar is
shown for TCM of non-transfected LM6 or H2M, because its OD was set to 1.
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