HEP-12-1861.R1
Epigenetic regulation of miR-122 by PPARγ and HBX
SUPPORTING EXPERIMENTAL PROCEDURES
Quantification of miRNA expression. Total RNA was isolated using Trizol (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instruction. The miScript Reverse
Transcription kit and miScript SYBR Green PCR kit (Qiagen, Valencia, CA) were used
to perform real-time PCR. Primer assay for mature miR-122 were obtained from Qiagen.
For detection of pri-miR-122, Taqman pri-miRNA assay kit (Applied Biosystems) was
used according to the manufacturer’s protocol. The Taqman reaction was performed with
Taqman Fast Universal PCR Master Mix (Applied Biosystem, Foster City, CA). Realtime PCR was performed in triplicates using Bio-rad CFX96 real-time system and data
analysis was performed using Bio-rad CFX Manager Software. The results were
calculated and normalized to an endogenous reference gene (RNU6B).
Plasmids and Luciferase Assays. The miR-122 promoter fragment (-595 to +165) was
cloned into the pGL4.12 vector (Promega) to produce the miR-122 promoter-Luc
reporter.
For luciferase assays, the cells were transfected with 1 μg of miR-122
promoter-Luciferase vector or pGL4.12 vector using Lipofectamine and plus reagents
(Invitrogen) in 6 well plates. Luciferase activity assays were performed 48 hours after
transfection using the Luciferase Reporter Assay System (Promega, Madison, WI).
Firefly luciferase activity was normalized to protein concentration.
siRNAs and Transfection. SUV39H1 siRNA duplexes were obtained from Qiagen and
PPARγ siRNA was purchased from Dharmacon RNAi Technologies (Lafayette, CO).
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HEP-12-1861.R1
Epigenetic regulation of miR-122 by PPARγ and HBX
The other siRNA oligonucleotide sequences used are as follow: HBX siRNA (sense: 5’GACCUUGAGGCAUACUUCAdTdT-3’
and
anti-sense:
5’-UGAAGUAUGCCUC
AAGGUCdTdT-3’), mock siRNA (sense: 5’-UUCUCCGAACGUGUCACGUdTdT-3’
and
anti-sense:
5’-ACGUGACACGUUC
GG
AGAAdTdT-3’),
as
previously
described(1). The cells were transfected with siRNA duplexes at the concentration of
100 nM using oligofectamine (Invitrogen). For transfection of primary hepatocyte, we
used Targefect-Hepatocyte (Targetingsystem, EI Cajon, CA) and the transfections were
carried out according to the manufacturer’s instructions.
miRNA microarray analysis. HepG2 cells were seeded at 1 X 106 cells/100 mm dish
and treated with 3 μM 5-Aza-CdR and 3 mM PBA for 48 hours. The culture media was
replaced every 24 hours containing same concentrations of 5-Aza-CdR and PBA. RNA
samples were isolated using the mirVana miRNA isolation Kit from Ambion (Ambion,
Austin, TX). miRNA microarray was carried out by LC sciences (LC Sciences, Houston,
TX). Total RNA samples labeled with Cy-3 and Cy-5 dye for dual sample analyses were
hybridized with the probe chip and signal measured after background normalization.
DNA pull down assay. The DNA pull down assay was performed according to a
previous report with minor modification(2). HepG2 cells were treated with 3 μM 5-AzaCdR and 3 mM PBA for 48 hours and then lysed by sonication in HKMG buffer (10 mM
HEPES, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, and 0.5% NP-40) with
protease inhibitors. Cell extracts were precleared with streptavidin-agarose beads for 1
hour, and then the supernatant was incubated with 1 μg of biotinylated double-stranded
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Epigenetic regulation of miR-122 by PPARγ and HBX
HEP-12-1861.R1
oligonucleotide and 10 μg poly (dI-dC) at 4°C for overnight.
The oligonucleotide
sequences
DR1);
are:
5’-Biotin-TGACCGGTGACTC-3’
(first
5’-Biotin-
AGGTGACCAGGTCA-3’ (DR2); and 5’-Biotin-TGGCCTAAGGTCG-3’ (second DR1).
The biotinylated oligonucleotides and their complementary strands were annealed in TEN
buffers. DNA-bound protein was collected by incubation with streptavidin-agarose beads
at 4°C for 1 hour. The agarose beads were washed four times with HKMG buffer and the
samples were boiled in SDS-sample buffer prior to SDS-PAGE and Western blotting
with specific antibodies.
Western blotting. The cells were lysed in a RIPA buffer (50 mM Tris-HCl pH 8.0, 150
mM NaCl, 5 mM EDTA and 1% NP-40 with protease inhibitor). After boiling for 5
minutes in the protein loading buffer with 2-mercaptoethanol, the samples were separated
on a SDS-PAGE gel and then transferred onto the nitrocellulose membrane (BioRad,
Hurcules, CA). The membranes were blocked in PBS-T containing 5% non-fat milk for
1 hour. The blots were incubated with different primary antibodies in PBS-T containing
5% non-fat milk at 4°C overnight. Following three times wash with PBS-T, membranes
were incubated with IRDye800 conjugated secondary antibodies for 1 hour. Blots were
washed with PBS-T and developed using LI-COR Odyssey Imaging system.
Immunopreipitation. The cells were resuspended in the lysis buffer (50 mM Tris-HCl
pH 7.4, 1% NP-40, 150 mM NaCl, 5 mM EDTA) containing complete protease inhibitor
tablet (Roche). Cells were lysed by quick freezing in liquid nitrogen and thawing in ice.
Cell debris was pelleted at 14,000 rpm for 15 min and lysates were precleared with 30 μl
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Epigenetic regulation of miR-122 by PPARγ and HBX
HEP-12-1861.R1
protein A/G-plus agarose beads (Santa Cruz Biotechnology) by rotation at 4°C for 1 hour.
The samples were collected by centrifugation at 5,000 rpm at 4°C for 5 min. The
supernatants were incubated with primary antibody at 4°C for overnight with rotation,
then incubated with 30 μl protein A/G-plus agarose beads for 2 hours. The precipitates
were washed five times with the wash solution (50 mM Tris-HCl pH7.6, 150 mM NaCl,
1 mM EDTA and 0.1% NP-40) and then suspended in 40 μl 2X SDS-PAGE sample
buffer. After boiling for 5 min, the samples were subjected to SDS-PAGE and Western
blotting with specific antibodies.
Chromatin immunoprecipitation (ChIP).
The assay was performed using a
Simplechip Enzymatic Chromatin IP kit (Cell Signaling Technology) according to the
manufacturer’s instructions. Immunoprecipitations were performed using 5 μg of antiPPARγ antibody, anti-acetyl histone antibody, anti-dimethyl histone H3K9 antibody and
anti-tri-methyl H3K9 antibody. Rabbit IgG antibody was used as a negative control.
ChIP primers were designed to amplify the different sites (DR1, DR2) in miR-122
promoter
region.
The
primers
are
as
follows:
for
1st
DR1,
5’-
GCAGATAAGGAGGAGCTTCA-3’ and 5’-AAGAGTCACCGGTCACAGGA-3’; for
DR2, 5’-TGTGACCGGTGACTCTTCTG-3’ and 5’-TGGAAAGTCCATTCCTCTGC-3’;
and
for
2nd
DR1,
5’-TGGACTTTCCAATCTTGCTG-3’
TCTCCCCTCTCCCTTTATGG-3’.
4
and
5’-
HEP-12-1861.R1
Epigenetic regulation of miR-122 by PPARγ and HBX
SUPPORTING REFERENCES
1.
Xie HY, Cheng J, Xing CY, Wang JJ, Su R, Wei XY, Zhou L, et al. Evaluation of
hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after
knockdown of HBx. Hepatobiliary Pancreat Dis Int 2011;10:295-302.
2.
Lu D, Han C, Wu T. Microsomal prostaglandin E synthase-1 inhibits PTEN and
promotes experimental cholangiocarcinogenesis and tumor progression. Gastroenterology
2011;140:2084-2094.
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HEP-12-1861.R1
Epigenetic regulation of miR-122 by PPARγ and HBX
SUPPORTING FIGURE LEGENDS
Supporting Figure S1.
5-Aza-CdR/PBA treatment does not alter the levels of
HNF4α, C/EBPα, and other related signaling molecules. HepG2 cells were treated
with 3 uM 5-Aza-CdR and 3 mM PBA for 48 hours and the cell lysates were obtained for
SDS-PAGE and Western blotting analysis using indicated antibodies.
(Panel A)
Representative western blots for HNF4α, C/EBPα and related molecules. (Panel B)
Representative western blots for other signaling molecules.
Supporting Figure S2. Representative western blots showing baseline levels of PPARγ,
RXRα, N-CoR, SMRT, SUV39H1, and H3K9 di-methyl and tri-methyl histone in human
primary hepatocytes (HH) and HCC cell lines (HepG2, Huh7 and Hep3B).
Supporting Figure S3. Subcellular localization of HBX and PPARγ in HepG2 cells
transiently transfected with HBX expression vector or control vector. The cells were
treated with 15-d-PGJ2 or DMSO for 24 hours. Cytosolic and nuclear fractions were
obtained for Western blotting using indicated antibodies. Whereas HBX is detected in
both cytoplasmic and nuclear fractions, PPARγ is detected only in nuclear fraction.
Loading controls include β-tubulin for cytoplasmic protein and PARP for nuclear protein.
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