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Supplementary Information
Supplementary Materials and Methods
Immunohistochemsitry and in situ hybridization. Consecutive tissue sections were used
for immunohistochemsitry and in situ hybridization. In situ hybridization experiments were
performed
according
to
the
protocol
provided
by
Exiqon
(Vedbaek,
Denmark)
(http://www.exiqon.com/uploads/LNA_52-_FFPE_miRNA_in_situ_protocol.pdf). Hybridization
was carried out at 45oC overnight with a miRCURY LNA™ miR-133b detection probe (50 nM)
(Exiqon). The same reaction with Scramble-miR (Exiqon) was performed as negative control.
RT-PCR and real-time PCR quantification. For the detection of pre-miRNA, cDNA was
synthesized from total RNA (50 ng/10 μl reaction) at 60oC for 50 min by using ThermoScript™
Reverse Transcriptase (Invitrogen, Carlsbad, CA). cDNA was diluted 5 fold and 2 μl of the
diluted cDNA mixture was used for PCR. PCR amplification was performed using the following
protocol with HotStarTaq® DNA Polymerase (Qiagen, Hilden, Germany): 95oC, 10 min; 95oC,
15 sec, 70oC, 15 sec, 55 cycles. The amplicons were separated by 8% polyacrylamide gel
electrophoresis. The oligonucleotides used in this study are listed in Supplementary Table S3.
LNA primer sets and miRCURY LNA™ microRNA PCR System (Exiqon) were used
according to the manufacturer’s instructions for the quantification of the expression of miR-133b
by reverse transcription and real-time PCR. Briefly, total RNA (10 ng) of each sample was
reverse transcribed in a 10 μl reaction. cDNA was diluted 4 fold and 4 μl of the diluted cDNA
mixture was used for real-time PCR using the LightCycler® 2.0 platform (Roche, Indianapolis,
IN) in the following procedure: 95oC, 10 min; 95oC, 10 sec, 60oC, 20 sec, 60 cycles. Parallel
reactions for the quantification of U6 RNA of all samples were also performed. The relative
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expression levels of miR-133b were determined by the following formula from the 2-
⊿ ⊿ CT
method (Livak and Schmittgen, 2001) with modifications:
-[(CT,133b – CT,U6)sample i – (CT,133b – CT,U6)sample m]
yi = E133b
yi is the relative miR-133b level of sample i. E133b is the mean PCR efficiency of miR-133b. PCR
efficiency of each reaction is determined on raw fluorescence data according to the reported
method (Tichopad et al., 2003). CT,133b is the threshold cycle of miR-133b amplification. CT,U6 is
the threshold cycle of U6 amplification. Sample m is the sample of normal cervical tissues which
has a median expression level of miR-133b. In the above formula, the relative miR-133b level of
sample m equals to 1.
The quantification of relative levels of miR-127 was similarly performed as described above.
For the quantification of relative levels of MST2 mRNA by reverse transcription and real-time
PCR, beta-actin gene was used as a reference gene.
Construction of stable cell lines. A sponge sequence containing multiple miRNA binding
sites was designed as described (Ebert et al., 2007). The sponge sequence containing four miR133b binding sites (5'-TAGCTGGTTCTCGGGACCAAA-3') was first cloned into pCMVpuromycin vector. The recombinant plasmid and empty vector were transfected into C33A cells
to generate C33A-miR-133b-sponge and C33A-NC respectively. Stable cell lines were selected
by using 1 μg/ml of puromycin and pooled.
In addition, a 349-bp human miR-133b gene fragment amplified from human genomic DNA
was cloned into pcDNA3.1-neomycin plasmid and sequence confirmed. The recombinant
plasmid and empty vector were transfected into CaSki cells to generate CaSki-miR-133b and
CaSki-NC respectively. Stable cell lines were selected by using 800 μg/ml of neomycin and
pooled. The miR-133b gene was also subcloned into pCMV-puromycin vector. The recombinant
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plasmid and empty vector were transfected into SiHa and HK-2 cells to generate SiHa-miR-133b
and HK-2-miR-133b and SiHa-NC and HK-2-NC, respectively. Stable cell lines were selected by
using 3 μg /ml of puromycin and pooled. The detailed information about all cell lines used in this
study is listed in Supplementary Table S2.
The gene fragment of miR-133b was also subcloned into pAdTrack-CMV vector for the
preparation of adnenoviruses expressing miR-133b.
Western blot assay. Total lysates (20 μg) were separated by 12% SDS-PAGE and electroblotted onto PVDF membranes. The PVDF membranes were blocked with 5% milk TBST for 2
h at room temperature. Reaction with primary antibody was carried out at 4°C overnight with
gentle shaking. After washing extensively with TBST, secondary antibody conjugated with HRP
was added and the reaction was carried out for 1 h at room temperature. SuperSignal ® West Pico
Chemiluminescent Substrate (Pierce, Rockford, IL) was used for the detection. Western blot
results were scanned and quantified by NIH ImageJ software.
The pcDNA3-EGFP-CDC42 and pcDNA3-EGFP-RHOA plasmids containing constitutively
active isoforms of CDC42 and RHOA were purchased from Addgene (Cambridge, MA). For the
investigation of the regulation of ATK1 activity by CDC42 and RHOA, the above plasmids or
the control plasmid (pcDNA3-EGFP) was transfected into CaSki-miR-133b cells and cell lysates
were prepared after 48 h transfection for Western blot assays.
Preparation of artificial miRNAs and siRNAs. Artificial miRNAs and siRNAs were
prepared by in vitro transcription as described previously (Boutz et al., 2007). The
oligonucleotides used for the preparation of miRNAs and siRNAs are listed in Supplementary
Table S3. Cel-miR-67 was used as the negative control miRNA. Renilla luciferase siRNA was
used as the negative control siRNA.
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SiRNA and miRNA inhibitor transfection. One day before transfection, cells (2×105/well)
were plated into a 6-well plate. SiRNA or hairpin miRNA inhibitor (Dharmacon, Lafayette, CO)
(100 nM) was transfected into cells using Lipofectamine™ 2000 (Invitrogen). Cell lysates were
prepared 48 h post-transfection.
Luciferase reporter assay. The 3′-UTR of human MST2 was amplified using PCR and
cloned into pRL-SV40 vector. The mutation of MST2 3′-UTR sequence was undertaken using
the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Luciferase reporter
assays were performed strictly according to previously published protocols (Boutz et al., 2007).
HPV detection. DNA from paraffin-embedded tissue sections was purified according to
the protocol of the Waldman lab (http://waldman.ucsf.edu/Protocols/paraffin.html). For the
detection of HPV by PCR, a High-risk HPV DNA (13 subtypes) Real-Time PCR Diagnostic kit
(Hybribio, Guangdong, China) was used, which can detect 13 HPV subtypes including HPV 16,
18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Twenty nanogram of purified DNA of each
sample was used for PCR and the reactions were performed according to the manufacturer’s
instructions. Samples with a CT value less than 40 is regarded as HPV-positive.
The HPVpan Detection Kit (Zhongshan Goldbridge Biotechnology, Beijing, China) was
used for further confirmation of HPV-negative carcinomas. The reactions were carried out
according to manufacturer's instructions. Briefly, tissue sections were deparaffinized in xylene
and washed with 100% ethanol. Then, samples were digested with pepsin at 37°C for 30 min and
dehydrated in graded ethanol. After denaturation at 95°C for 5 min, hybridization was performed
at 37°C for 2 h. Mouse anti-biotin antibody and HRP-conjugated anti-mouse IgG were
sequentially added to tissue sections. Color was developed with DAB. For ISH detection of HPV,
MaxArray™ HPV Control Cell Slides (Invitrogen) were used as negative and positive controls.
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Supplementary References
Boutz PL, Chawla G, Stoilov P, Black DL. (2007). MicroRNAs regulate the expression of the
alternative splicing factor nPTB during muscle development. Genes Dev., 21: 71-84.
Ebert MS, Neilson JR, Sharp PA. (2007). MicroRNA sponges: competitive inhibitors of small
RNAs in mammalian cells. Nat. Methods, 4: 721-726.
Livak KJ, Schmittgen TD. (2001). Analysis of relative gene expression data using real-time
⊿⊿CT
quantitative PCR and the 2-
method. Methods, 25: 402-408.
Tichopad A, Dilger M, Schwarz G, Pfaffl MW. (2003). Standardized determination of real-time
PCR efficiency from a single reaction set-up. Nucleic Acids Res., 31: e122.
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Supplementary Figure Legends
Supplementary Figure S1. MiR-133b is up-regulated in cervical carcinoma tissues. (a) The
overlap of three miRNA profiling results includes miR-133a, miR-133b and miR-127. (b) The
sequences of hsa-miR-133a, hsa-miR-133b, pre-hsa-miR-133a-1, pre-hsa-miR-133a-2 and prehsa-miR-133b. Identical nucleotides are shaded in the pre-miRNA sequences. (c) Discrimination
of the two isoforms by pre-miRNA amplification. N: no template (negative control); P:
pcDNA3.1-miR-133b or pcDNA3.1-miR-133a as the template (positive control); 1 - 6: cDNA
from carcinoma samples as templates; M: 20 bp DNA Ladder Marker, from top to bottom: 500,
400, 300, 200, 180, 160, 140, 120, 100, 80, 60, 40, 20 bp. (d) Expression of pre-miR-133b in
normal cervical tissues and cervical carcinoma tissues. The relative levels of pre-miR-133b were
determined by ImageJ software and normalized to the levels of U6 RNA. (e) Quantification of
miR-133b levels in normal cervical tissues (N1-N6) and cervical carcinomas (T1-T8). (f)
Quantification of miR-127 levels in normal cervical tissues (N1-N6) and cervical carcinomas
(T1-T8). The red line in (d) or (e) indicates the median level of miR-133b or miR-127 in normal
tissues.
Supplementary Figure S2. MiR-133b enhances in vitro colony formation, while miR-127 has
little detectable effects on colony formation. (a) Quantification of miR-133b levels in CaSki cells
transiently transfected with empty vector (NC) or miR-133b-expression plasmid. (b)
Quantification of miR-127 levels in CaSki cells transiently transfected with empty vector (NC)
or miR-127-expression plasmid. (c) Number of colonies formed by CaSki cells transiently
transfected with empty vector (NC), miR-127 or miR-133b-expression plasmid.
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Supplementary Figure S3. The expression of miR-133b is up-regulated in HPV-negative
cervical carcinomas. (a) Detection of HPV infection by real-time PCR. (b) PCR products of the
reactions in (a) were separated by 8% polyacrylamide gel. 1: negative control; 2: positive control;
3 - 5: three HPV-negative carcinoma samples; 6 - 9: four HPV-positive carcinoma samples; M:
20 bp DNA Ladder Marker, from top to bottom: 500, 400, 300, 200, 180, 160, 140, 120, 100, 80,
60, 40, 20 bp. (c) ISH results of HPV in cervical carcinoma tissues. The HPV-negative cells,
HPV 6/11-positive cells, HPV 16/18-positive cells and HPV 31/33/35-positive cells were from
Invitrogen’s MaxArray™ HPV Control Cell Slides and were used as negative and positive
controls for ISH analysis. (d) ISH results with MiRCURY LNA™ miR-133b detection probe or
Scramble-miR in normal tissues and HPV-negative carcinoma tissues. H&E: hematoxylin and
eosin staining; Ki-67 IHC: immunohistochemistry with anti-Ki-67 antibody. Scale bars in (c) and
(d) = 100 μm. (e) Quantification of the expression levels of miR-133b in HPV-negative
carcinomas by real-time RT-PCR.
Supplementary Figure S4. MiR-133b promotes tumorigenesis. (a) Design of the miR-133b
sponge. Transcription of the sponge sequence is driven by the CMV promoter and terminated by
the BGH polyadenylation signal. (b) Confirmation of the expression of the sponge sequence
containing four miR-133b binding sites in C33A stable cell lines by RT-PCR. 1: control C33A
cell lines constructed with empty vector (C33A-NC); 2: C33A cell lines constructed with the
sponge expression vector (C33A-miR-133b-sponge). (c) Relative expression levels of miR-133b
in CaSki and SiHa stable cell lines quantified by real-time RT-PCR. The expression levels of
miR-133b in CaSki and SiHa control stable cell lines were normalized to 1. (d) C33A stable cell
colonies formed in soft agar assays. (e) CaSki and SiHa stable cell colonies formed in soft agar
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assays. (f) Relative expression levels of miR-133b in HK-2 stable cell lines quantified by realtime RT-PCR. The expression levels of miR-133b in HK-2 control stable cell lines were
normalized to 1. (g) Colony numbers formed by HK-2 stable cell lines in soft agar assays. (h)
Tumors formed by HK-2-NC and HK-2-miR-133b stable cells two months after inoculation. In
(c), (e), (f), (g) and (h), NC represents stable cell lines constructed using empty vectors as
negative control, while miR-133b represents stable cell lines constructed using miR-133b
expressing plasmids.
Supplementary Figure S5. MiR-133b promotes metastasis. (a) Mouse lungs 8 weeks after
intravenous injection of SiHa stable cell lines. (b) Hematoxylin and eosin stained tissue sections
showing metastatic foci formed in mouse lungs 4, 6 and 8 weeks after intravenous injection of
SiHa stable cells. (c) Ki-67 stained tissue sections showing metastatic foci formed in mouse
lungs 4, 6 and 8 weeks after intravenous injection of SiHa stable cells. For 4- and 6-week
experiments, n = 3. Arrows indicate metastatic foci. Scale bar = 100 μm (4 and 6 weeks). Scale
bar = 200 μm (8 weeks).
Supplementary Figure S6. MiR-133b targets CDC42 and RHOA. (a) Immunoblotting of
CDC42 and RHOA in CaSki cells infected with miR-133b expression (Ad-miR-133b) or empty
vector (Ad-NC) adenoviruses. Cell lysates were prepared after 48 h infection. (b)
Immunoblotting using anti-CDC42 antibody. CaSki-miR-133b cells were transfected with
pcDNA3-EGFP or pcDNA3-EGFP-CDC42 plasmid. 1: pcDNA3-EGFP; 2: pcDNA3-EGFPCDC42. (c) Immunoblotting using anti-RHOA antibody. CaSki-miR-133b cells were transfected
with pcDNA3-EGFP or pcDNA3-EGFP-RHOA plasmid. 1: pcDNA3-EGFP; 2: pcDNA3-EGFP-
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RHOA. (d) CDC42 and RHOA inhibit AKT1 activity. CaSki-miR-133b cells were transfected
with CDC42-expressing, or RHOA-expressing, or EGFP-expressing plasmid. 1: pcDNA3-EGFP;
2: pcDNA3-EGFP-CDC42 + pcDNA3-EGFP-RHOA; 3: pcDNA3-EGFP-CDC42; 4: pcDNA3EGFP-RHOA.
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Supplementary Table S2.
Information about the cell lines used in this study.
Cell line
HPV status
HK-2
C33A
CaSki
HeLa
SiHa
HK-2-NC
HPV-16 E6/E7
HPV negative
HPV-16
HPV-18
HPV-16
HPV-16 E6/E7
Tumor
forming
No
Yes
Yes
Yes
Yes
No
HK-2-miR-133b
HPV-16 E6/E7
Yes
C33A-NC
HPV negative
Yes
C33A-miR133b-sponge
HPV negative
Yes
CaSki-NC
HPV-16
Yes
CaSki-miR-133b
HPV-16
Yes
SiHa-NC
HPV-16
Yes
SiHa-miR-133b
HPV-16
Yes
#
Stable cell line information
HK-2 cells carrying pCMV-puromycin
vector#
HK-2 cells carrying pCMV-puromycinmiR-133b which expresses the microRNA
miR-133b#
C33A cells carrying pCMV-puromycin
vector#
C33A cells carrying pCMV-puromycinmiR-133b-sponge which expresses a
‘sponge sequence’ of four miR-133b
binding sites#
CaSki cells carrying pcDNA 3.1-neomycin
vector#
CaSki cells carrying pcDNA 3.1-neomycinmiR-133b which expresses the microRNA
miR-133b#
SiHa cells carrying pCMV-puromycin
vector#
SiHa cells carrying pCMV-puromycin-miR133b which expresses the microRNA miR133b#
: This work.
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Supplementary Table S3.
The oligonucleotides used in this study.
Sequence
Amplification of 5' GCGGCGGTGCTTTGCTAGAGCTGGTAAAA 3'
pre-miR-133a
5' CGGCGGAGCTACAGCTGGTTGAAGGG 3'
Amplification of 5' CGCGGCTGCTCTGGCTGGTCAAACG 3'
pre-miR-133b
5' CGGCGGTCAGGAAGACGGACTTGGTT 3'
Amplification of 5′ CTGACAGGATCCGTAAGAGGACATTCTGGACAAGGCAAGC 3′
miR-133b gene
5′ CGCACGAATTCATTCCTGGGAGCATAAGAATATGGTGAAA 3′
T7-promoter
5′-TAATACGACTCACTATAGGGAGACAGG-3′
containing primer
Preparation
of 5′-AATTGGTCCCCTTCAACCAGCCCTGTCTC-3′
artificial
miR- 5′-TAGCTGGTTGAAGGGGACCAACCTGTCTC-3′
133b
Preparation
of 5′-AATCACAACCTCCTAGAAAGAGTACCTGTCTC-3′
artificial cel-miR- 5′-TCTACTCTTTCTAGGAGGTTGTGACCTGTCTC-3′
67
Preparation
of 5′-AATAAATAAGAAGAGGCCGCGCCTGTCTC-3′
Renilla luciferase 5′-AACGCGGCCTCTTCTTATTTACCTGTCTC-3′
siRNA
Preparation
of 5′-AAGCCCATATGTTGTAAAGTACCTGTCTC-3′
Mst2 siRNA
5′-AATACTTTACAACATATGGGCCCTGTCTC-3′
Amplification of 5'ACAGCACTGAAGGGCTTTATGTTAC 3'
Mst2 3′-UTR
5' GGTGGCACAGTGAAATTTATTTGTC 3'
Mutation of Mst2 5' TTTATATGATTGCATTGGCAGCAGTCATTTCCTAAGCTAC 3'
3′-UTR
5' GTAGCTTAGGAAATGACTGCTGCCAATGCAATCATATAAA 3'
Amplification of 5' CCTGTTAGATCTGTTCACGCTTTCCTCCACAAT 3'
miR-127 gene
5' ACGATGTCTAGACCCTACTGCCTGAAGAACTGC 3'
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