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Supporting Information
Patient samples
The 41 hepatocellular carcinoma tissues and their corresponding noncancerous
hepatic tissues utilized in this study were obtained with informed consent from
patients who underwent radical resections in the Eastern Hepatobiliary Surgery
Hospital (Second Military Medical University, Shanghai, China). The study was
performed in accordance with the guidelines of the Institutional Review Board of the
Liver Cancer Institute.
Cell culture
SMMC-7721 and HepG2 cells were obtained from the China Center for Type Culture
Collection (Wuhan, China). The cells were grown in Dulbecco’s Modified Eagle
Medium (Gibco BRL) with 10% fetal bovine serum (Gibco BRL) and were
maintained in an atmosphere of 5% CO2 in a humidified 37 °C incubator.
Reverse transcription reactions and quantitative real-time PCR
Total RNAs were extracted using the Trizol reagent (Takara, Dalian, China). The
first-strand cDNA was generated using the Reverse Transcription System Kit
(Promega Corporation, Madison WI). Stem-loop reverse transcription reactions for
the mature microRNAs and U6 primers were performed as previously described (1).
U6 RNA was employed as a miRNA internal control. Real-time PCR was performed
using a standard SYBR-Green PCR kit protocol in a StepOne Plus system (Applied
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Biosystems, Foster City, CA). β-actin was employed as an endogenous control to
normalize the amount of total mRNA in each sample. The real-time PCR reactions
were performed in triplicate. The relative expression of RNAs was calculated using
the comparative Ct method. The primer sequences are presented in Supporting Table
1.
DNA methylation analysis
Genomic DNA was extracted from human HCC tissues and adjacent noncancerous
hepatic tissues with the Axygen genomic DNA purification kit (Axygen
Biotechnology, Hangzhou, China). Genomic DNA (0.5 ug) was treated with sodium
bisulfite with the Zymo EZ DNA Methylation Gold kit (Zymo Research, Orange, CA)
according to the manufacturer’s instructions. Modified genomic DNA was then
amplified with primers specific to the mir-200a promoter by PCR (2). The primers
sequences are presented in Supporting Table 1. Bisulfite genomic sequencing PCR
products were gel-extracted, subcloned into pMD-19T Vectors (Takara), and
transformed into Escherichia coli. Candidate plasmid clones were sequenced by
Invitrogen, Ltd (Shanghai, China).
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed according to the
Acetyl-Histone H3 Immunoprecipitation Assay Kit (Millipore, USA). ChIP-derived
DNA was quantified using qRT-PCR with SYBR Green incorporation (Applied
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Biosystems). The primers specific for the mir-200a promoter or the p21WAF/Cip1
promoter (3) are listed in Supporting Table 1.
Small interfering RNA (siRNA) synthesis
The siRNAs specifically targeting HDAC4 and control siRNA (the sequences are
depicted in Supporting Table 1) were synthesized by GenePharma (Shanghai, China)
as described (3). The siRNAs for Sp1 were purchased from Santa Cruz Biotech (Santa
Cruz, CA).
Transient transfection
Transfections were performed using the Lipofectamine 2000 kit (Invitrogen)
according to the manufacturer’s instructions. The double-stranded microRNAs
mimics, single-stranded microRNAs inhibitors and their respective negative control
RNAs (GenePharma) were introduced into cells at a final concentration of 50 nM.
The transfected cells were harvested at 24, 48, or 72 hours after transfection.
Construction of vectors
The complementary DNA encoding HDAC4 was PCR-amplified by the Pfu Ultra II
Fusion HS DNA Polymerase (Stratagene, Agilent Technologies, Palo Alto, CA) and
was subcloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The promoter
region of the mir-200a, from -965 to +193 bp upstream of the transcription start site
(4), was PCR-amplified by TaKaRa LA Taq (Takara) and was subcloned into the
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pGL3 basic firefly luciferase reporter. The p21WAF/Cip1 promoter subcloned into the
same vector was used as a positive control. The two pGL3 constructs containing the
mir-200a promoter with various point mutations in the Sp1 binding sites were
synthesized using a QuikChange Site-Directed Mutagenesis kit (Stratagene). The 3’
untranslated regions (3’-UTR) of HDAC4 mRNA containing the two intact miR-200a
recognition sequences were PCR-amplified and subcloned into a pMIR-REPORT
vector (Applied Biosystems) immediately downstream of the luciferase gene. SIP1
3’-UTR (1.1kb) subcloned into the same vector was used as a positive control. All of
the primer sequences are presented in Supporting Table 1.
Luciferase reporter assay
The pcDNA3.1-HDAC4, HDAC4 siRNA or miRNA were cotransfected with
luciferase reporter plasmids into cultured cells by Lipofectamine-mediated gene
transfer. Each sample was cotransfected with the pRL-TK plasmid, which expressed
renilla luciferase to monitor transfection efficiency (Promega). The relative luciferase
activity was normalized with renilla luciferase activity.
Western blot analysis
Total cell lysates were prepared in a 1× sodium dodecyl sulfate buffer. Identical
quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred onto polyvinylidene fluoride membranes. After
incubation with antibodies specific for HDAC4 (ProteinTech Group, Chicago, USA),
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acetyl-histone H3 (Millipore) or β-actin (Sigma-Aldrich, USA), the blots were
incubated with IRdye 800-conjugated goat anti-rabbit IgG and IRdye 700-conjugated
goat anti-mouse IgG and were detected using an Odyssey infrared scanner (Li-Cor,
Lincoln, NE).
Generation of cells stably transfected with the miR-200a
Recombinant lentiviruses containing the pre-hsa-miR-200a or the control were
purchased from GeneChem (Shanghai, China). To generate the stable cell line, 4 x 105
cells were transfected with 2×106 transducing units of lentiviruses and were selected
with 2 μg/ml puromycin for two weeks.
Measurement of cell proliferation
A total of approximately 3.0 × 103 HCC cells were plated in 96-well plates. After 24 h
of culture, cell proliferation was assessed using the Cell Counting Kit-8 (Beyotime,
Jiangsu, China) according to the manufacturer’s protocol. All of the experiments were
performed in triplicate. The cell proliferation curves were plotted using the
absorbance at each time point.
Transwell assays
SMMC-7721 or HepG2 cells were plated in medium without serum in the upper
chamber of a transwell (24-well insert, 8-mm pore size, Millipore). The invasive
activity of the two cells lines was analyzed, as described (5).
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In vivo tumorigenesis assay
Cells from the miR-200a and the control vector stable expression cell lines (1.0×107)
were implanted subcutaneously into the flanks of nude mice. We analyzed primary
tumor growth by measuring the tumor length (L) and width (W), and we calculated
tumor volume according to the equation V=0.4×LW2. The animal studies were
approved by the Institutional Animal Care and Use Committee of the Second Military
Medical University, Shanghai, China.
Statistical analysis
Differences between the expression levels of the miR-200a and HDAC4 in HCC
patients were compared through the Wilcoxon signed-rank test. The relationship of
miR-200a and HDAC4 mRNA expression was analyzed by Pearson’s correlation.
Bisulfite DNA sequencing results were compared by the Wilcoxon rank-sum test.
Others comparisons were determined by Student’s t-test. All P values were two-sided
and obtained using the SPSS 18.0 software package (SPSS, Chicago, IL, USA).
Differences were defined as statistically significant for p-values ≤ 0.05.
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