1
Supporting Information
MicroRNA-195
Suppresses
Angiogenesis
and
Metastasis
of
Hepatocellular Carcinoma by Inhibiting the Expression of VEGF,
VAV2 and CDC42
Ruizhi Wang, Na Zhao, Siwen Li, Jian-Hong Fang, Mei-Xian Chen, Jine Yang,
Wei-Hua Jia, Yunfei Yuan and Shi-Mei Zhuang
INVENTORY
A)
Supporting Materials and Methods pages 2-11
B)
Supporting References
page 12
C)
Supporting Figure legends
pages 13-21
2
Supporting Materials and Methods
Vector construction
The retroviral expression vectors for miR-195 were generated by cloning the genomic
fragments (737 bp) encompassing the corresponding miR-195 precursor and its 5’and 3’-flanking sequences into pRetroX-Tight-Pur (Clontech, Palo Alto, CA, USA).
The resultant vectors were designated as pRetroX-miR-195.
Retroviral expression vectors for firefly luciferase (pMir-vec-LUC) were constructed
by cloning the coding sequence of firefly luciferase (1668 bp) downstream of the
CMV promoter in pMir-vec (kindly provided by Dr. Reuven Agami1), which contains
a blasticidin resistance gene.
pc3-Gab-VEGF, pc3-Gab-VAV2 and pc3-Gab-CDC42 that expressed VEGF, VAV2
and CDC42, respectively, were created by inserting the coding sequences of VEGF
(726 bp), VAV2 (2636 bp) or CDC42 (576 bp) into pc3-Gab,2 which was based on
pcDNA3.0 (Invitrogen Corp., NY, USA).
For the construction of pGL3cm-VEGF-3'UTR-Wt, pGL3cm-VAV2-3'UTR-Wt,
pGL3cm-CDC42-3'UTR-Wt, the wild-type 3'UTR segments of human VEGF (455
bp), VAV2 (525 bp) or CDC42 (596 bp) mRNA that contained putative binding sites
for miR-195 were inserted downstream of the stop codon of firefly luciferase in
pGL3cm,3 which was based upon the pGL3-control (Promega, Madison, WI, USA).
3
The
plasmids
pGL3cm-VEGF-3'UTR-Mut,
pGL3cm-VAV2-3'UTR-Mut
or
pGL3cm-CDC42-3'UTR-Mut, which carried the mutated sequences in the
complementary sites of the seed region of miR-195, were produced by site-specific
mutagenesis based on pGL3cm-VEGF-3'UTR-Wt, pGL3cm-VAV2-3'UTR-Wt or
pGL3cm-CDC42-3'UTR-Wt. The sequences of the primers used for PCR are
provided in Supporting Table 2.
Packaging of retroviral expression vectors and infection of target cells
HEK293T cells were co-transfected with 1.6 μg of retroviral expression vector and
1.6 μg of the pCL-Ampho packing plasmid (Imgenex, San Diego, CA, USA) in a
6-cm dish and were cultured in 5 ml complete medium for 48 h, followed by
collection of the supernatant. The retroviral supernatant was passed through a
0.45-μm filter immediately after collection and stored at -80 °C. Target cells were
grown to 40% confluence in a 6-well plate, refreshed with 3 ml of retroviral
supernatant supplemented with 10 μg/ml polybrene (Sigma, St. Louis, MO, USA) and
centrifuged at 600g (60 min, 30 °C). After centrifugation, the supernatant was
replaced by fresh DMEM medium with 10% FBS. The infections were repeated 3-4
times at 12 h intervals, and stable lines were then selected.
Establishment of QGY-7703 subline that stably expressed miR-195 and luciferase
The QGY-7703 subline that stably expressed miR-195 and luciferase and the control
line were established as follows: first, QGY-7703 cells were infected with a retrovirus
4
that expressed the tetracycline (Tet)-controlled transactivator (pRetroX-Tet-Off
Advanced) and were then subjected to selection with 500 μg/ml of G418 (Clontech).
Subsequently, the selected cells were infected with retroviruses that expressed
hsa-miR-195 (pRetroX-miR-195) or the empty vector (pRetroX-Tight-Pur) under the
control of a Tet-regulated promoter, followed by selection with 500 μg/ml of G418
and 0.5 μg/ml of puromycin (Clontech) in the presence of doxycycline (Dox, 0.1
μg/ml, Clontech), a tetracycline analogue. The resulting stable lines (QGY-miR-195
and QGY-control) were infected further with the retrovirus that expressed firefly
luciferase (pMir-vec-LUC) and were selected with 0.5 μg/ml of blasticidin (Sigma).
The final stable cell lines (QGY-miR-195-LUC and QGY-control-LUC) were
maintained in DMEM that was supplemented with 10% FBS, 100 μg/ml of G418,
0.25 μg/ml of puromycin, 0.25 μg/ml of blasticidin and 0.1 μg/ml of Dox.
Analysis of gene expression
The primers used for semi-quantitative RT-PCR are listed in Supporting Table 2.
qPCR analysis of miR-195 expression was performed on a LightCycler 480 (Roche
Diagnostics, Germany) with a TaqMan MicroRNA Assay kit (Applied Biosystems,
Foster City, CA, USA). All reactions were run in triplicate. The cycle threshold (Ct)
values did not differ by more than 0.5 among the triplicates. The miR-195 level was
normalized to RNU6 to permit calculations of the 2-△△Ct value.
The following antibodies were used for immunoblotting: mouse monoclonal
5
antibodies (mAb) against VEGF (cat. sc-7269, Santa Cruz Biotechnology, Santa Cruz,
CA, USA), ERK1/2 and phospho-T202/Y204-ERK1/2 (cat. 610030 and cat. 612358,
BD Biosciences, Franklin Lakes, NJ, USA), Rac1 (cat. 05-389, Millipore, Billerica,
MA, USA), Rb (cat. 9309, Cell Signaling Technology (CST, Beverly, MA, USA),
β-actin and GAPDH (cat. BM0627 and cat. BM1623, Boster, Wuhan, PR China);
rabbit mAb against VAV2, CDC42 and phospho-Ser807/811-Rb (cat. 2848, cat. 2462
and cat. 9308, CST); and rabbit polyclonal antibodies (pAb) against VEGFR2 and
phospho-Tyr1175-VEGFR2 (cat. 2479 and cat. 2478s, CST).
The microvessel density (MVD) and expression of VEGF, VAV2, CDC42 and Ki-67
in tissues were analyzed by immunohistochemical staining (IHC) as follows:
formalin-fixed, paraffin-embedded tissues were cut into 5μm sections, placed on
poly-lysine-coated slides, 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). The sections were incubated at 4°C overnight with mouse mAb against human
CD34 (cat. sc-52312, Santa Cruz) and Ki-67 (cat. ZM-0166, Zhongshan, PR China),
or rabbit pAb against VAV2 (cat. SAB4503068, Sigma) or CDC42 (cat. BA2442,
Boster), or rabbit mAb against VEGF or mouse CD146 (cat. ZA-0509 and cat.
ZA-0539, Zhongshan). Immunostaining was performed with the ChemMate DAKO
EnVision Detection Kit for Peroxidase/DAB/Rabbit/Mouse (code K 5007,
DakoCytomation, Glostrup, Denmark), which resulted in a brown-colored precipitate
6
at the antigen site. Subsequently, the sections were counterstained with hematoxylin
(Zymed Laboratories, South San Francisco, CA, USA) and mounted with
non-aqueous mounting medium. All runs included a no primary antibody control.
The microvessel density (MVD) in human tissues was evaluated based on CD34
staining and was determined from the five most intensely vascularized areas of each
section at a magnification of 200X. MVD in the xenograft tumor tissues was
evaluated by CD146 staining and was quantified in the three most intensely
vascularized areas or the whole areas of small tumors under light microscopy at a
magnification of 100X. Any discrete cluster or single cell that was stained for CD34
or CD146 was counted as one microvessel. The average value of the vessel count per
field was used as the final MVD value for each case.
The proliferation rate in human HCC tissues was evaluated on the basis of Ki-67
staining and was determined from five random areas of each section at a
magnification of 400X. A total of 1,000 to 2,000 tumor cells were counted, and the
proportion of positively stained cells was used to represent the proliferation rate of
each specimen.
VEGF, VAV2 or CDC42 expression in the tissues was evaluated under a light
microscope at a magnification of 400X. For each specimen, five images of
representative areas were acquired, and a total of 1,000 to 2,000 tumor cells were
7
counted. IHC scoring was performed according to a modified Histo-score (H-score),
which included a semiquantitative assessment of both the fraction of positive cells and
the intensity of staining. The intensity score was defined as no staining (0), weak (1),
moderate (2) or strong (3) staining. The fraction score was based on the proportion of
positively stained cells (0-100%). The intensity and fraction scores were multiplied to
obtain an H-score that ranged from 0 to 3 and represented the expression level of each
protein.
Preparation of tumor-cell conditioned medium (TCM)
QGY-7703 or MHCC-97H cells were reverse transfected with RNA mimics in 6-well
plates and QGY-7703 cells were reverse transfected with RNA inhibitors or siRNAs.
The medium was removed thirty-six hours after the transfections. The cells were
washed with 1X PBS three times and were cultured in 2 ml (for QGY-7703) or 1 ml
(for MHCC-97H) of Serum-Free Medium for endothelial cells (SFM) for 12 h.
Afterwards, the TCM was centrifuged sequentially at 500g to remove the detached
cells and at 12,000g to discard cell debris (4 °C, 10 min each). Aliquots of TCM were
stored at -80 °C until used. Each corresponding well was subsequently trypsinized and
the numbers of live cells were counted to permit the appropriate correction of TCM
loading for equivalent cell numbers.
HUVEC recruitment and capillary tube formation assays
For in vitro endothelial recruitment assays, 24-well Boyden chambers with 8-μm pore
8
size polycarbonate membranes (Corning, NY) were used. HUVECs (5×104) were
resuspended in 100 μl of SFM and seeded in the upper compartments of the chambers,
and tumor cells were placed in the lower compartments in 600 μl of SFM. The
co-cultured tumor cells were non-transfected or transfected with miRNA mimics or
inhibitors for 36 h and refreshed with SFM before the recruitment experiments.
For capillary tube formation assays, HUVECs (1.5×104) were grown in the absence or
presence of 100% TCM for 6 h at 37°C in a 96-well plate coated with Matrigel
(3432-005-01, R&D Systems, MN, USA). The formation of capillary-like structures
was captured under a light microscope. The branch points of the formed tubes, which
represented the degree of angiogenesis in vitro, were scanned and quantitated in five
low-power fields (100X).
In vitro tumor cell migration, invasion and growth assays
Tumor cell migration and invasion were analyzed in 24-well Boyden chambers with
8-μm pore size polycarbonate membranes (Corning). For invasion assays, the
membranes were coated with 60 μg of Matrigel to form matrix barriers. QGY-7703,
MHCC-97H, MHCC-97L, Huh-7, SMMC-7721 and HCT-116 cells were analyzed in
the migration assay, and QGY-7703 and MHCC-97H cells were analyzed in the
invasion assay. All cell lines were reverse transfected with RNA mimics and
QGY-7703 cells were reverse transfected with RNA inhibitors or siRNAs. For both
the migration and invasion assays, the cells (5×104 QGY-7703 or SMMC-7721; 1×105
9
MHCC-97H, MHCC-97L, Huh-7 or HCT-116) were resuspended in 100 μl
serum-free DMEM at 36 h post-transfection and were added to the upper
compartments of the chambers, and the lower compartments were filled with 600 μl
of DMEM with 10% FBS. After an incubation at 37°C for 12 h (QGY-7703,
SMMC-7721, Huh7 and HCT-116) or 20 h (MHCC-97H and MHCC-97L), the cells
remaining on the upper surfaces of the membrane were removed. The cells on the
lower surfaces of the membrane were fixed, stained with crystal violet and counted
under a light microscope.
To analyze cell growth, non-transfected, NC- or miR-195-transfected cells
(QGY-7703, MHCC-97H, MHCC-97L, Huh-7, SMMC-7721 or HCT-116) were
grown in 24-well plates, and the cell numbers were counted every 24 h for 5 days
post-transfection.
Nude mouse xenograft studies and in vivo imaging
Bioluminescence imaging was performed according to the manufacturer's instructions.
Briefly, mice were injected intraperitoneally with a luciferin solution (150 mg/kg
body weight, Cat. XR-1001, Xenogen, Alameda, CA, USA) that was allowed to
distribute in conscious animals for approximately 5 min. Next, the mice were placed
into an anesthesia box (with 2.5-3.5% isofluorane). After the mice were fully
anesthetized, they were transferred to the imaging chamber, and images were taken.
The bioluminescence values were calculated by measuring the number of
10
photons/second.
For the ex vivo imaging of individual organs, mice were sacrificed at 5 min
post-administration of luciferin, and the organs were immediately collected and
imaged. For imaging of intrahepatic metastases, the left hepatic lobe with the primary
tumor was removed and the remaining lobes of the liver were imaged. The
bioluminescence values were calculated by measuring the photons/second for each
organ.
After ex vivo imaging, the primary tumors, livers and lungs were dissected, fixed in
formalin, embedded in paraffin, and sectioned.
GST pull-down assay
GST or GST-PAK fusion proteins were purified from Escherichia coli that had been
transformed with pGEX-4T3-GST or pGEX-4T3-GST-PAK, as described previously.4
Briefly, bacteria expressing GST or GST-PAK fusion proteins were cultured overnight,
collected and sonicated in a bacterial lysis buffer supplemented with protease
inhibitors.
GST
or
GST-PAK
fusion
proteins
were
purified
with
glutathione-Sepharose 4B beads (27-4574-01, Amersham Pharmacia, Piscataway, NJ).
For the GST pull-down assay, QGY-7703 cells were trypsinized and resuspended at
48 h post-transfection in a eukaryotic cell lysis buffer supplemented with protease
11
inhibitors and were lysed on ice. The lysates were collected and incubated with
glutathione-Sepharose 4B bead-immobilized GST or GST-PAK for 1 h at 4 °C. After
the incubation, the beads were washed extensively with eukaryotic cell lysis buffer,
three times at 5 min per wash, and the bead-bound proteins were solubilized in SDS
buffer and analyzed by immunoblotting. For each sample, 1/20 of lysate was saved
and subjected to total Rac1 detection as an internal control.
12
Supporting References
1.
Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, et al.
A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in
testicular germ cell tumors. Cell 2006;124:1169-81.
2.
Xu T, Zhu Y, Xiong Y, Ge YY, Yun JP, Zhuang SM. MicroRNA-195 suppresses
tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma
cells. Hepatology 2009;50:113-121.
3.
Su H, Yang JR, Xu T, Huang J, Xu L, Yuan Y, Zhuang SM. MicroRNA-101,
down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses
tumorigenicity. Cancer Res 2009;69:1135-1142.
4.
Benard V, Bohl BP, Bokoch GM. Characterization of rac and cdc42 activation in
chemoattractant-stimulated human neutrophils using a novel assay for active
GTPases. J Biol Chem 1999;274:13198-13204.
13
Supporting Figure Legends
Supporting Fig. 1. miR-195 represses tumor angiogenesis in vitro. (A) An endothelial
recruitment assay revealed the suppressive effect of miR-195 on the HCC
cell-promoted migration of endothelial cells (ECs). HUVECs were seeded in the
upper compartments of transwell chambers, and MHCC-97H cells that were
non-transfected (panel 2) or transfected with NC (panel 3) or miR-195 (panel 4) were
seeded in the lower compartments. Co-cultures were conducted for 12 h in
Serum-Free Medium for endothelial cells (SFM). SFM (panel 1), in which no tumor
cells were placed in the bottom chamber, was used as a control. (B) Restoration of
miR-195 inhibited the HCC cell-promoted tube formation of ECs. HUVECs were
cultured for 6 h in the presence of SFM (panel 1) or 100% tumor-cell conditioned
medium (TCM) from MHCC-97H cells, which were non-transfected (panel 2) or
transfected with NC (panel 3) or miR-195 duplex (panel 4). Scale bar, 100 µm. *, P <
0.05; **, P < 0.01; ***, P < 0.001.
Supporting Fig. 2. miR-195 suppresses tumor migration and invasion in vitro. (A, B)
Restoration of miR-195 inhibited the migration and invasion of HCC cells.
MHCC-97H cells that were non-transfected (panel 1) or transfected with NC (panel 2)
or miR-195 duplex (panel 3) were added to transwell chambers without (A) or with
(B) Matrigel coatings and were incubated for 20 h, followed by staining with crystal
violet. Scale bar, 100 µm. **, P < 0.01.
14
Supporting Fig. 3. Analysis of miR-195 expression in QGY-miR-195-LUC and the
control line QGY-control-LUC by qPCR. QGY-miR-195-LUC cells were cultured
with (+) or without (-) Dox for 48 h; QGY-control-LUC cells were cultured without
Dox. The miR-195 level was normalized to RNU6 expression. No error bar is shown
for the control because the normalized miR-195 level was set to 1. *, P < 0.05.
Supporting Fig. 4. miR-195 suppresses tumor growth and angiogenesis in vivo. (A, B)
miR-195 suppressed tumor growth in vivo. Orthotopic xenograft tumor growth (A)
was monitored by in vivo bioluminescent luciferase detection at the indicated days
after tumor implantation. Tumor volumes (B) were measured on the 50th day after
tumor implantation. Tumors developed in the implanted sites in 6/8 mice in the
miR-195-off group and 5/8 in the miR-195-on group. The dissected tumors were
measured for length (L) and width (W) with calipers, and the tumor volumes (V) were
calculated according to the formula: V = (L×W2) ×0.5. (C) Orthotopic xenograft
tumors from miR-195-on mice displayed lower microvessel densities (MVD). On the
50th day after implantation, tumor tissues from the miR-195-off or -on groups were
stained for CD146. Scale bar, 50 µm. (D, E) Restoration of miR-195 expression
inhibited tumor metastasis in the orthotopic xenograft model. The intrahepatic (D) and
pulmonary metastases (E) were detected by ex vivo bioluminescent imaging on the
50th day after implantation. For the imaging of intrahepatic metastases (D), the left
hepatic lobe with primary tumors was removed before imaging. For (A-E),
QGY-miR-195-LUC cells were inoculated under the capsule of the left hepatic lobe of
15
BALB/c nude mice. All mice were fed with Dox for the first 10 days prior to division
into the miR-195-on and -off groups, followed by Dox withdrawal in the miR-195-on
group and continued Dox treatment in the miR-195-off mice for another 40 days. For
(A), (D) and (E), luciferase signals were quantified by measuring the photons/second.
*, P < 0.05; **, P < 0.01.
Supporting Fig. 5. In vivo bioluminescent detection of primary xenograft tumors on
the 40th day after implantation. Tumors developed in the implanted sites in 9/15 mice
in the miR-195-off group and 8/14 in the miR-195-on group. Luciferase signals were
quantified by measuring the photons/second. The central horizontal line represents the
mean value; the error bars represent the SEM.
Supporting Fig. 6. Restoration of miR-195 expression inhibits tumor metastasis in an
orthotopic xenograft model. (A, B) miR-195 expression suppressed intrahepatic
metastasis. (C, D) miR-195 expression repressed pulmonary metastasis. The
intrahepatic and pulmonary metastases were detected by ex vivo bioluminescent
imaging (A, C) and histopathological analysis (B, D). On the 40th day after
implantation, mice were sacrificed 5 min after the injection of luciferin, and the
organs were collected and imaged ex vivo. For imaging of the intrahepatic metastases,
the left hepatic lobe with primary tumors was removed before imaging. After ex vivo
imaging, the livers and lungs were serially sectioned and subjected to
Hematoxylin-eosin-staining and histopathological analysis. The central horizontal line,
16
mean value; error bar, SEM.
Supporting Fig. 7. VEGF is a direct target of miR-195. (A) miR-195 and its putative
binding sequence in the 3'UTR of VEGF. Mutations were generated in the
complementary site that binds to the seed region of miR-195. (B) Restoration of
miR-195 decreased the level of secreted VEGF. TCM from MHCC-97H cells that
were non-transfected or transfected with NC or miR-195 duplex were analyzed by
ELISA. (C) Knockdown of endogenous miR-195 enhanced the cellular VEGF level.
QGY-7703 cells that were non-transfected (lane 1) or transfected with anti-NC (lane 2)
or anti-miR-195 (lane 3) for 48 h were analyzed by immunoblotting. GAPDH was
used as an internal control. The results were reproduced in three independent
experiments and representative immunoblots are shown. (D) Restoration of miR-195
expression reduced VEGF levels in orthotopic xenograft tumors. (E) The level of
miR-195 correlated inversely with VEGF expression in human HCC tissues. For (D)
and (E), a brown signal was considered to be a positive VEGF stain. Images were
captured at a magnification of 400X. Scale bar, 50 µm. *, P < 0.05; ***, P < 0.001.
Supporting Fig. 8. miR-195 exerts its anti-angiogenic function by inhibiting
VEGFR2-signaling in ECs. (A) Restoration of miR-195 in HCC cells attenuated
VEGFR2 signaling in ECs. Twenty-four hours after seeding, HUVECs were grown in
SFM for 12 h and were then cultured for 15 min in the presence of SFM (lane 1) or
100% TCM from MHCC-97H cells that were non-transfected (lane 2) or transfected
17
with NC (lane 3) or miR-195 (lane 4), followed by immunoblotting for
phosphor-Tyr1175-VEGFR2, VEGFR2, phosphor-T202/Y204-ERK1/2 and ERK1/2
expression. β-actin was used as an internal control. (B) Antagonism of miR-195 in
HCC cells enhanced VEGFR2 signaling in ECs. Twenty-four hours after seeding,
HUVECs were grown in SFM for 12 h, then were cultured for 15 min in the presence
of SFM (lane 1) or 100% TCM from QGY-7703 cells that were non-transfected (lane
2) or transfected with anti-NC (lane 3) or anti-miR-195 (lane 4), followed by
immunoblotting as in (A). The results in both (A) and (B) were reproduced in three
independent experiments, and representative immunoblots are shown.
Supporting Fig. 9. Knockdown of VEGF inhibits HCC cell-promoted angiogenesis in
vitro. (A) Cellular levels of VEGF were reduced by si-VEGF transfection. QGY-7703
cells that were non-transfected (lane 1) or transfected with NC (lane 2), miR-195
(lane 3) or si-VEGF (lane 4) were analyzed by RT-PCR. Hypoxanthine-guanosine
phosphoribosyl transferase (hPRT) was amplified in the same reactions as an internal
control. The results were reproduced in three independent experiments and a
representative image is shown. (B) Knockdown of VEGF inhibited the HCC
cell-promoted migration of ECs. HUVECs were seeded in the upper compartments of
transwell chambers, and QGY-7703 cells transfected with NC (bar 1) or si-VEGF
(bar 2) were seeded in the lower compartments. Co-cultures were conducted for 12 h
in SFM. (C) Knockdown of VEGF repressed the HCC cell-promoted tube formation
of ECs. HUVECs were cultured for 6 h in the presence of 100% TCM from
18
QGY-7703 cells, which were transfected with NC (bar 1) or si-VEGF (bar 2). The
capillary tube formation of HUVECs was assessed subsequently. *, P < 0.05; **, P <
0.01.
Supporting Fig. 10. Over-expression of VEGF attenuates the anti-angiogenic effect of
miR-195. (A) Immunoblotting showed the efficiency of VEGF over-expression.
Forty-eight hours after transfections with pc3-Gab or pc3-Gab-VEGF, QGY-7703
cells were analyzed by immunoblotting. GAPDH was used as an internal control. (B)
Introduction of VEGF antagonized the anti-angiogenic effect of miR-195. HUVECs
were cultured in TCM derived from QGY-7703 cells that were co-transfected with the
following RNA duplex/expression plasmid combinations: NC/empty vector (panel 1),
miR-195/empty vector (panel 2), NC/VEGF (panel 3) or miR-195/VEGF (panel 4).
The capillary tube formation of HUVECs was assessed subsequently. The results in
both (A) and (B) were reproduced in three independent experiments, and
representative images are shown. Scale bar, 100 µm.
Supporting Fig. 11. VAV2 and CDC42 are direct targets of miR-195. (A) miR-195 and
its putative binding sequences in the 3'UTR of VAV2 and CDC42. Mutations were
generated in the complementary sites that bind to the seed region of miR-195. (B)
Expression of miR-195 reduced the protein levels of cellular VAV2 and CDC42.
MHCC-97H cells that were non-transfected (lane 1) or transfected with NC (lane 2)
or miR-195 (lane 3) for 48 h were analyzed by immunoblotting. (C) Knockdown of
19
endogenous miR-195 enhanced the expression of VAV2 and CDC42. HCC cells that
were non-transfected (lane 1) or transfected with anti-NC (lane 2) or anti-miR-195
(lane 3) for 48 h were analyzed by immunoblotting. For (B) and (C), GAPDH was
used as an internal control. The results were reproduced in three independent
experiments and representative immunoblots are shown. (D) Restoration of miR-195
expression reduced VAV2 and CDC42 expression in orthotopic xenograft tumors. (E)
miR-195 expression correlated inversely with VAV2 or CDC42 expression in human
HCC tissues. For (D) and (E), a brown signal was considered to be a positive VAV2 or
CDC42 stain. The images were captured at a magnification of 400X. Scale bar, 50 µm.
***, P < 0.001.
Supporting Fig. 12. Knockdown of VAV2 and CDC42 inhibits tumor migration in
vitro. (A) Cellular mRNA levels of VAV2 and CDC42 were reduced by si-VAV2 and
si-CDC42 transfection. QGY-7703 cells that were non-transfected (lane 1) or
transfected with NC (lane 2), miR-195 (lane 3), si-VAV2 (lane 4) or si-CDC42 (lane
5) were analyzed by RT-PCR. GAPDH was used as an internal control. The results
were reproduced in three independent experiments and a representative image is
shown. (A) Knockdown of VAV2 and CDC42 inhibited the migration of HCC cells.
QGY-7703 cells transfected with NC (bar 1), si-VAV2 (bar 2) or siCDC42 (bar 3)
were added to transwell chambers and incubated for 12 h, followed by staining with
crystal violet. ***, P < 0.001.
20
Supporting Fig. 13. Over-expression of VAV2 or CDC42 attenuated the anti-migration
effect of miR-195. (A) Immunoblotting showed the efficiencies of VAV2 and CDC42
over-expression. Forty-eight hours after transfections with pc3-Gab, pc3-Gab-VAV2
or pc3-Gab-CDC42, QGY-7703 cells were analyzed by immunoblotting. GAPDH was
used as an internal control. The results were reproduced in three independent
experiments and a representative immunoblot is shown. (B) Introduction of VAV2 or
CDC42 antagonized the anti-migration effect of miR-195. QGY-7703 cells that were
co-transfected with the following RNA duplex/expression plasmid combinations:
NC/empty vector (bar 1), miR-195/empty vector (bar 2), NC/VAV2 (bar 3),
miR-195/VAV2 (bar 4), NC/CDC42 (bar 5), miR-195/CDC42 (bar 6) were added to
transwell chambers and incubated for 12 h, followed by staining with crystal violet.
**, P < 0.01; ***, P < 0.001.
Supporting Fig. 14. Overexpression of miR-195 inhibited lamellipodia formation in
QGY-7703 cells. QGY-7703 cells that were non-transfected or transfected with NC,
miR-195, si-VAV2 or si-CDC42 for 48 h were added to Matrigel-coated plates and
incubated for 1 h, followed by staining for filamentous actin. Representative images
of the lamellipodia (arrows) in cells are shown. Arrows indicate the mature
lamellipodias. Scale bar, 10 µm.
Supporting Fig. 15. The level of miR-195 correlated inversely with the proliferation
rate of tumor cells in human HCC tissues. This correlation was analyzed in the 90
21
HCC tissues described in Fig. 5C. The median value of the proportion of
Ki-67-positive tumor cells in 90 cases was chosen as the cut-off point for separating
the low- (n = 45) from the high-proliferation-rate (n = 45) tumors. **, P < 0.01.
Supporting Fig. 16. Effect of miR-195 over-expression on the growth of QGY-7703
(A) and MHCC-97H cells (B). Cells that were non-transfected or transfected with NC
or miR-195 duplex were grown in 24-well plates, and the cell numbers were counted
every 24 h for 5 days after the transfections. *, P < 0.05; ***, P < 0.001.
Supporting Fig. 17. Effect of miR-195 over-expression on the growth and migration
of MHCC-97L (A), Huh-7 (B), SMMC-7721 (C) and HCT-116 cells (D). Cells that
were non-transfected or transfected with NC or miR-195 duplex were analyzed for
the growth (panel 1) and migration (panel 2). *, P < 0.05; **, P < 0.01; ***, P <
0.001.
Supporting Fig. 18. Effect of miR-195 over-expression on Rb signaling in MHCC97L
and QGY-7703 cells. Forty-eight hours after transfections with the indicated RNA
duplexes, cells were subjected to immunoblotting or RT-PCR analysis. pRb, total Rb
protein; ppRb, phosphorylated pRb. β-actin, internal control.
Supporting Fig. 19. miR-195 affects various phenotypes of cancer cells by regulating
different signaling pathways.