Supporting Materials
Materials and Methods
Immunohistochemistry. The HCC tissue and normal liver tissue microarrays were
obtained from the Xi'an Aomei Biotechnology Co., Ltd. (Xi'an, China). These microarrays
were composed of 40 HCC tissues, 8 normal liver and 12 paratumor liver tissues, which
included duplicate core biopsies (1 mm in diameter) from fixed, paraffin-embedded tumors.
Immunohistochemical staining of samples were performed as previously reported1 and the
primary antibody of rabbit anti-YAP (Proteintech Group, USA) was used. The extents of
cytosolic and nuclear staining were considered in the scoring. The percentage of
immunoreactivity in tumor cells or hepatocytes was graded as: 0 (<10%); 1, low (10%-30%);
2, intermediate (30%-50%); 3, high (>50%). Categorization of immunostaining intensity was
performed by three independent observers.
Cell Lines and Cell Culture. Hepatoma cell lines HepG2, HepG2-P, HepG2-X and
HepG2.2.15 (a hepatoma HepG2 cell line integrated full-length HBV DNA), and human
embryonic kidney cell line 293T were maintained in Dulbecco’s modified Eagle’s medium
(Gibco, Grand Island, NY).2, 3 H7402, H7402-P and H7402-X cell lines2 were cultured in
RPMI Medium 1640 (Gibco) supplemented with 10% fetal calf serum (FCS), 100 U/ml
penicillin, and 100 mg/ml streptomycin in 5% CO2 at 37℃.
Cell Transfection. The cells were cultured in a 6-well or 24-well plate for 24 h and then
were transfected with plasmid or siRNA. All transfections were performed using
1
Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s
protocol. The pSilencer-X (pSi-HBx) produce small interfering RNAs (siRNAs) targeting
HBx mRNA and pSilencer-control (pSi-Con) as negative control were used.1 The plasmid
pCH-9/3091 containing the complete HBV genome has been described previously.4 SiRNA
oligonucleotides, which target YAP5, 6and CREB7, and a non-specific scrambled control (NC)
are synthesized by RiboBio (Guangzhou, China). The siRNA duplexes sequences are all listed
in Supporting Table 2.
RNA Extraction, Reverse-Transcription, and Quantitative Real-Time Polymerase Chain
Reaction (qRT-PCR). Total RNA was extracted from cells (or liver tissues from
HBx-transgenic (HBx-Tg) mice and patient tissues) using Trizol reagent (Invitrogen).
First-strand cDNA was synthesized as reported before.7 To analyze miRNA-375 expression,
total RNA was polyadenylated by poly (A) polymerase (Ambion, Austin, TX, USA) as
described previously.8 Reverse transcription was performed using poly (A)-tailed total RNA
and reverse transcription primer with ImPro-II Reverse Transcriptase (Promega, Madison, WI,
USA) according to the manufacturer’s protocol. QRT-PCR was performed by a Bio-Rad
sequence detection system according to the manufacturer’s instructions using double-stranded
DNA-specific SYBR GreenPremix Ex TaqTM II Kit (TaKaRa Bio, Dalian, China).
Experiments were conducted in duplicate in three independent assays. Relative transcriptional
folds were calculated as 2-ΔΔ Ct.9 GAPDH was used as an internal control for normalization.
U6 was used as an internal control to normalize miRNA-375 levels. All the primers used are
listed in Supporting Table 2. The HBV DNA in the supernatants of HepG2 cells transfected
2
with pCH-9/3091 was extracted using the Blood & Cell Culture DNA kit (QIAGEN,
Germany) following the manufacturer’s instructions. Real-time PCR was used to quantify
HBV DNA copies according to a diagnostic kit for quantification of HBV DNA (Da An Gene,
Guangzhou, China) in a Bio-Rad sequence detection system.10
Nuclear Protein Extraction and Western Blot Analysis. The nuclear extraction assay was
performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce，USA)
following the manufacturer’s instructions. The western blot protocol was described
previously.2 Primary antibodies used were rabbit anti-YAP (Proteintech), rabbit anti-AFP
(Proteintech), mouse anti-HBx (Abcam, Cambridge, UK), rabbit anti-CREB (Santa Cruz, CA,
USA), rabbit anti-Histone H3 (Signalway Antibody, USA) and mouse anti-β-actin
(Sigma-Aldrich, St. Louis, MO). The experiment was performed according to the previous
reported protocol.2 All experiments were repeated three times.
Plasmid Construction. The 5'-flanking region (nucleotides -1420 to +115) of YAP was
amplified by PCR from the genomic DNA of HepG2 using specific primers and was cloned
into the upstream of the pGL3-Basic vector (Promega) via KpnI and XhoI sites. The resulting
plasmid was sequenced and named pGL3-1536. To construct various lengths luciferase
reporter plasmids of YAP, the regions (-1000/+115, -604/+115, -354/+115, -232/+115,
-62/+115, -41/+115) of YAP were amplified by PCR from the pGL3-1536 and were inserted
into the pGL3-Basic vector to generate pGL3-1116, pGL3-720, pGL3-470, pGL3-348,
pGL3-178, pGL3-157, respectively. Mutant construct of pGL3-348, named as pGL3-348
3
MUT, which carried a substitution of two nucleotides(ATAT instead of wild-type ACGT)
within the binding sites of CREB, was carried out using overlapping extension PCR. 11 All
primers are listed in Supporting Table 2.
Luciferase Reporter Gene Assay. Luciferase reporter gene assay was performed using the
Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s
instructions. Cells were transferred into 24-well plates at 3 × 104 cells per well. After 24 h, the
cells were transiently co-transfected with 0.1 μg/well of pRL-TK plasmid (Promega)
containing the Renilla luciferase gene used for internal normalization, and various constructs
containing different lengths of the YAP 5'-flanking region or pGL3-Basic. The luciferase
activities were measured as previously described.12 All experiments were performed at least
three times.
Chromatin Immunoprecipitation (ChIP). ChIP assays were performed in HepG2-X cells
transfected siRNA control (NC) or CREB siRNA (si-CREB) according to the manufacturer’s
protocol (Epigentek Group Inc, Brooklyn, NY) as reported previously.7,
13
The promoter
region of the YAP including the CREB sites was amplified from the immunoprecipitated
DNA samples with the specific primers. All primers are listed in Supporting Table 2. All
experiments were performed at least three times.
Electrophoretic Mobility Shift Assay (EMSA). The EMSA protocol was described
previously.14 Briefly, nuclear extracts were prepared from HepG2-X cells. Probes were
4
generated by annealing single strand oligonucleotides containing the CREB consensus
sequence (-231/-200) of YAP promoter and labeling the ends with [γ-32P] ATP using T4
polynucleotide kinase (TaKaRa Bio). The oligonucleotide sequences (-231/-200) are listed in
Supporting Table 2. EMSA was performed with 1 µg of nuclear extract in binding buffer (20
mM Hepes, pH 7.9, 0.05 mM EDTA, pH 8.0, 50 mM KCl, 1 mM MgCl2, 0.5 mM DTT and
5% glycerol) containing 1 µg of poly (dI-dC). 30-fold excess of unlabeled DNA competitor or
1 µg anti-IgG antibody (Santa Cruz) or 1 µg anti-HBx antibody (Abcam) or 1 µg anti-CREB
antibody (Santa Cruz) was incubated with nuclear extracts on ice for 30 min before labeled
probes were added into the binding buffer. Samples were incubated on ice for 1 h and then
separated by electrophoresis on 6% non-denaturing polyacrylamide gel, and then the gel was
dried and subjected to autoradiography.
Analysis of Cell Proliferation. HepG2/H7402, HepG2-X/H7402-X cells were seeded onto
96 well plates (1000 cells/well) for 24h before transfection and 3-(4,5-dimethylthiazol-2-yl)
-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell proliferation every day
from the first day until the third day after transfection. The protocol was described
previously.12 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay was carried out using the
Cell-Light TM EdU imaging detecting kit according to the manufacturer’s instructions
(RiboBio).
Analysis of Colony Formation. For clonogenicity analysis, 48 h after transfection, 1000
viable transfected cells (HepG2/H7402, HepG2-X/H7402-X) were placed in 6-well plates and
5
maintained in complete medium for 2 weeks. Colonies were fixed with methanol and stained
with methylene blue.
Animal Transplantation. Nude mice were housed and treated according to guidelines
established by the National Institutes of Health Guide for the Care and Use of Laboratory
Animals. We conducted the animal transplantation according to the Declaration of Helsinki.
Tumor transplantation in nude mice was performed. Briefly, HepG2-X cells were harvested
and re-suspended at 2 × 107 per ml with sterile phosphate-buffered saline. Groups of
4-week-old female BALB/c athymic nude mice (Experiment Animal Center of Peking, China)
(each group, n=5) were subcutaneously injected at the shoulder with 0.2 ml of the cell
suspensions. Tumor growth was measured after 10 days from injection and then every 5 days.
Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers
and calculated with the formula (L × W2) × 0.5. After 30 days, tumor-bearing mice and
controls were sacrificed, and the tumors were excised and measured.
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