Supporting Material and Methods
MiRNA Transfections and RNA Interference Experiments. MiR-148a mimics (AM17100,
ID PM10263) and antagonists (AM17000, ID AM10263), negative control miRNA mimics
(AM17110) and antagonists (AM17010), and mouse Dnmt1 siRNAs (ID s65071 and s65072)
were all purchased from Ambion. Negative control siRNAs were from Qiagen. HCC cell line
transfections were carried out 24 h after seeding at a density of 4 × 105 cells in 35-mm-diameter
dishes using the TransFectin lipid reagent (Bio-Rad Laboratories). Cells were incubated for 5 h
with the transfection mix containing 100 nM of miRNA mimics or antagonists and 5 µL of
TransFectin in a 1 mL total volume of OptiMEM (Invitrogen) free of antibiotics and serum.
Transfections of MFH primary cultures by miRNA mimics or antagonists were performed as
described above for HCC cells on the fourth day following cell sorting (MFH D4), whereas
transfections by siRNAs (100 nM) were performed the second day after cell sorting (MFH D2),
when Dnmt1 expression was still significant.
Total RNA Isolation. After washing the cells with PBS, miRNAs and mRNAs were extracted
using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Total RNAs
from human HCC biopsy samples were extracted using the same protocol. Before RNA
extraction, mouse fetal and adult livers were collected in cold PBS and physically dissociated,
and blood cells were lysed using the Red Blood Cell Lysing Buffer (Sigma) as recommended by
the manufacturer. Total RNAs were quantified using a NanoDrop 1000 spectrophotometer
(Thermo Scientific), and the RNA integrity was evaluated with an Agilent 2100 Bioanalyzer
(Agilent Technologies).
MiRNA Microarray. Total RNA samples (2,000 ng) were collected from MFHs at days 2, 4,
6, 8, and 10 following their isolation and the beginning of the induced differentiation process.
1
RNA labeling and hybridization were performed using a human microRNA microarray kit
(Agilent Technologies) according to the manufacturer’s protocol (Protocol for Use with Agilent
MicroRNA Microarrays Version 1.5). Hybridization signals were detected using a DNA
Microarray Scanner (Agilent Technologies), and the scanned images were analyzed using
Agilent Feature Extraction software (v.10.7.3.1; Agilent Technologies). All the microarray data
are shown in Supporting Table 1. We applied a
2-fold change in signal intensity to identify
the significant differences of miRNA expression in this study.
MiRNA and mRNA Real-Time Quantitative PCR. For miRNA analyses by real-time
quantitative polymerase chain reaction (RT-qPCR), 100 ng of total RNA was reverse-transcribed
using the Taqman miRNA Reverse Transcription Kit (Applied Biosystems). The expression
levels of mature miR-148a, miR-148b, miR-152, and control RNU6B were determined by RTqPCR with the Taqman Universal PCR Master Mix in a 7300 Real-Time PCR System from
Applied Biosystems. The PCR conditions were 50°C for 2 min and 95°C for 10 min, followed by
40 cycles of 95°C for 15 sec and 60°C for 1 min. Taqman probes from Applied Biosystems were
used to assess the expression of the 3 miRNAs, which are conserved between mouse and human
species: miR-148a (ID 000470), miR-148b (ID 000471), and miR-152 (ID 000475). The
expression levels of miRNAs were normalized with respect to the endogenous levels of RNU6B
(ID 001093). To determine mRNA expression, RNA samples were first treated with DNase
using the TURBO DNA-free kit (Ambion). Then, cDNAs were synthesized from 1 µg of total
RNA of each fraction using SuperScript III Reverse Transcriptase (Invitrogen) following the
manufacturer’s recommendations. Finally, SYBR Green quantitative PCRs were performed to
determine mRNA expression (Platinum SYBR Green qPCR SuperMix-UDG, Invitrogen). After
an initial denaturation at 95°C for 2 min, the thermal cycles were repeated 40 times as follows:
2
95°C for 15 sec and 60°C for 30 sec. The GAPDH housekeeping gene was used to normalize the
variations in the cDNA levels. The sequences of the primers used for gene amplification are
shown in Supporting Table 3.
Assessment of miR-148a Levels in the Serum of HCC Patients. The levels of circulating
miR-148a in serum were analyzed from 11 HCC patients with underlying HCV infection. The
isolation of extracellular miRNAs was performed using the miRNeasy Mini Kit (Qiagen) as
described above. Importantly, RNAs were extracted from the same volume of serum (200 µL)
for each sample. In addition, before phenol extraction, 0.1 nM of synthetic C. elegans miR-39
was added to each aliquot. C. elegans miR-39 expression was used as an invariant control from
200 µL of serum to normalize miR-148a expression data.
Luciferase Reporter Assay. The 3’-UTR reporter plasmids were all purchased from
GeneCopoeia, where the 3’-UTR sequences of DNMT1 (accession numbers NM_010066.3 and
NM_001379.1 for mouse and human, respectively) or c-MET (NM_008591.2 and NM_000245.2
for mouse and human, respectively) were cloned downstream of the firefly luciferase reporter
gene in the pEZX-MT01 vector. Renilla luciferase activity driven by a CMV promoter was used
for normalization. Site-directed mutagenesis for the construction of mutated 3’-UTR vectors was
performed in the seed sequences of mouse Dnmt1 and human DNMT1. PrimeSTAR Max DNA
Polymerase (Takara, Japan) was used for PCR amplification. Forward primer and reverse primer
sequences were as follows: mmu-Dnmt1-mut-Fw, ccacggtacatgatgtttttaaccctttgagcc; mmuDnmt1-mut-Rev,
catcatgtaccgtggggctctgggtgagagca;
caacagtacatgatgttgtgtttttaacatgtc;
hsa-DNMT1-mut-Rev,
hsa-DNMT1-mut-Fw,
catcatgtactgttggggattcctggtgccaga.
Simultaneous transfections were performed using 3’-UTR constructs (3 µg) and miRNA mimics
(100 mM) in 35-mm-diameter dishes following the experimental procedure described above.
3
Empty vectors and control miRNA mimics were used as negative controls. Cells were collected
24 h after transfection, and proteins were extracted on ice using the M-PER Mammalian Protein
Extraction Reagent (Thermo Scientific). The firefly-Renilla luciferase signals were assayed by
the Dual-Glo Luciferase Assay System (Promega) using the Envision system (Wallac) as
recommended by the manufacturer.
Immunoblotting. Protein extraction was performed using M-PER Mammalian Protein
Extraction Reagent (Thermo Scientific). Total protein (10 µg) was resolved by SDS-PAGE
(10%) and transferred to nitrocellulose membranes for 1 h. Membranes were incubated overnight
at 4°C with the following primary antibodies: anti-Dnmt1 (ab13537; abcam) and anti-Albumin
(Santa Cruz; sc-46293) using appropriate dilution: 1/1,500 and 1/2,000, respectively. Anti-βTubulin (Sigma-Aldrich) at 1/1,500 was used as loading control. Antigen–antibody complexes
were visualized by a chemiluminescence substrate procedure using the ECL Plus Western
blotting detection system (GE Healthcare) and scanned with the Fujifilm LAS-3000 imaging
system (Fujifilm).
DNA Methylation Analysis. After washing the cells with PBS, Genomic DNAs were
extracted using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich)
according to the manufacturer’s protocol. DNA was quantified using a NanoDrop 1000
spectrophotometer (Thermo Scientific). Combined bisulfite restriction analysis (COBRA) was
used for analyzing miR-148a and mouse albumin promoter methylation status. Briefly, genomic
DNA was treated with sodium bisulfite using an EpiTect Plus kit (QIAGEN) and applied to PCR,
followed by digestion with restriction enzymes. The primers used are summarized in Supporting
Table 4.
4
Periodic Acid-Schiff (PAS) Staining. MFHs or mature-induced hepatocytes were fixed on ice
for 5 min using glacial methanol. After 2 washes with PBS, PAS staining was performed using
Schiff reagent, periodic acid, and hematoxylin (PAS Staining System Kit, Sigma)
5-Aza Treatment. To inhibit the global methylation status of DNA, cells were treated with 1,
2.5, and 5 µM of 5-aza-2’-deoxycytidine (5-Aza; Sigma) for 5 days. The drug and medium were
replaced daily.
Apoptotic Activity. Hepa 1-6 and HepG2 cells (4 × 105 cells in a 35-mm-diameter dish) were
transfected with miR-148a mimics or controls as described above. Twenty-four hours after
transfection, cells were treated with the apoptosis inducer TRAIL at 50 ng/mL (PeproTech) or
left untreated. Cells were collected at the indicated times, and proteins were extracted with MPER (Thermo Scientific). Caspase-3/7 activity was assessed using the Apo-ONE Homogeneous
Caspase-3/7 Assay (Promega) at an excitation wavelength of 480 nm and an emission
wavelength of 520 nm using the Envision system (Wallac).
Cell Growth Assay. Twenty-four hours after miRNA mimic or antagonist transfection, cells
were seeded at 10,000 cells per well in 96-well plates, and cell viability was measured at the
indicated times using the Tetra Color One assay kit (Seikagaku, Japan) according to the
manufacturer’s instructions. The absorbance at 450 nm was measured using the Envision system
(Wallac).
Wound-Healing Assay. Hepa 1-6 cells were used for wound-healing assays. Twenty-four
hours after transfection with miR-148a mimics or antagonists, five wounds per well were made
by aspiration of the confluent cell layers using a plastic tip. Cells were washed twice with PBS to
remove the detached cells and debris and maintained in DMEM supplemented with HGF (50
5
ng/mL). The sizes of the wounds were observed and measured at the indicated times using the
ImageJ software (http://rsbweb.nih.gov/ij/).
Transwell Invasion Assay. The invasion ability of Hepa 1-6 cells was assessed in 24-well
Biocoat Matrigel invasion chambers with 8-µm pore size (BD Biosciences) according to the
manufacturer’s recommended protocol. The day following miRNA mimic transfection, Hepa 1-6
were trypsinized, and 50,000 cells were plated in the upper chamber with a serum-free medium.
The bottom chamber contained 10% FBS and HGF (100 ng/mL) as chemoattractants. Cells that
migrated through the membrane were fixed with methanol 48 h later and stained using the DiffQuik staining kit (SYSMEX). Invasive cells were automatically counted using the ImageJ
software, and the average number of cells per field was calculated.
Statistical Analysis. Experimental data are presented as the mean ± SD. Student’s t-test was
performed for the estimation of statistical significance between two groups of data. Equality of
variances was tested using an F-test. All the P-values were two-tailed. Correlation analysis
between miR-148a and DMNT1 expression level were assessed by calculating Spearman’s rank
coefficient. Statistical significance of the miR-148a levels among normal livers, HCCs, and
adjacent non-tumor tissues was assessed using the Mann-Whitney U-test. The miR-148a
expression differences in HCC liver biopsies between tissue pairs (tumors and their
corresponding adjacent non-tumor areas) were compared by the Wilcoxon signed-rank test. All
statistical assessments were performed using MedCalc software. A result was considered
statistically significant at P < 0.05. Experimental data and RT-qPCR profiles shown in this
article are representative of at least three independent experiments.
6
Supporting Table 1. Microarray data. (see attached Excel file)
7
Supporting Table 2. Clinical data from HCC patients, normal livers, and fetal livers.
Patient
number
Gender
Age
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
M
F
M
M
M
M
M
M
M
M
F
F
M
M
62
36
58
58
56
58
56
67
63
41
68
40
57
61
61
Virus
serology
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
HBV
16
17
18
M
M
F
44
60
51
HBV
HBV
HBV
HBV
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
M
F
M
M
M
M
M
M
M
M
F
M
M
M
F
66
77
70
71
63
55
52
69
62
65
62
62
66
59
72
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
HCV
Clinical history
Chronic hepatitis
Chronic hepatitis
Chronic hepatitis
Liver cirrhosis
Chronic hepatitis
Precirrhosis
Precirrhosis
Chronic hepatitis
Chronic hepatitis
Precirrhosis
Chronic hepatitis
Liver cirrhosis
Chronic hepatitis
Chronic hepatitis
Precirrhosis
8
Precirrhosis
Liver cirrhosis
Chronic hepatitis
Chronic hepatitis
Liver cirrhosis
Chronic hepatitis
Chronic hepatitis
Liver cirrhosis
Liver cirrhosis
Chronic hepatitis
Chronic hepatitis
Chronic hepatitis
Chronic hepatitis
Liver cirrhosis
Liver cirrhosis
Precirrhosis
Chronic hepatitis
Precirrhosis
34
35
36
37
38
39
F
F
M
M
F
F
69
61
65
68
57
74
HCV
HCV
HCV
HCV
HCV
HCV
Chronic hepatitis
Precirrhosis
Liver cirrhosis
Precirrhosis
Precirrhosis
Liver cirrhosis
N01
N02
N03
N04
N05
N06
N07
N08
N09
N10
N11
N12
N13
M
F
M
M
F
F
M
M
M
M
M
M
M
71
62
46
80
65
63
57
61
55
54
64
71
71
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
non B/C
F01
F02
F03
F04
F05
M
M/F
F
F
M
18-20 w
22-40 w
20 w
20 w
24 w
N/A
N/A
N/A
N/A
N/A
Leukemia
Colon cancer metastasis
Colon cancer metastasis
Neuroendocrine cancer metastasis
Leukemia
Gastric cancer metastasis
Colorectal cancer metastasis
Neuroendocrine cancer metastasis
Colon cancer metastasis
Colorectal cancer metastasis
Intracranial hemorrhage
Unknown
Unknown
Pool of 3 (18, 20, and 18 weeks)
Pool of 63
Single abortus
Single abortus
Single abortus
HBV and HCV-related HCC samples included tumor and non-tumor tissues for each patient.
9
Supporting Table 3. List of primers used for RT-qPCR.
Gene
Name
Sense
Antisense
glyceraldehyde 3-phosphatase
dehydrogenase
alpha-fetoprotein
albumin
glucose-6-phosphatase
tyrosine aminotransferase
cytokeratin 19
DNA methyltransferase 1
DNA methyltransferase 3a
DNA methyltransferase 3b
hepatocyte growth factor receptor
(HGFR)
cytochrome P450 17A1
(steroid 17-alpha-monooxygenase)
cytochrome P450 17A1
equivalent to human CYP3A4
cytochrome P450 1a2
cytochrome P450 3a41
cytochrome P450 3a44
acccagaagactgtggatgg
cacattgggggtaggaacac
gaagcaagccctgtgaactc
aggcgactatctccagcaaa
atgactttgggatccagtcg
gccaatcctggacagaacat
caccatgcaaaacctcaatg
cccaaagaaggatcctgtga
accaggccacctacaacaag
acttggtgattggtggaagc
cgatcagcagtctgtgcatt
ccgagaaatctgcagtgaca
cctcaacaaaatcagcagca
tggaaccagatgggaaagag
ttctgaaggtgccgcttact
caaggcgtgttctgtctcaa
cttgatgtctgcctcgttga
tgcttgttctgcacttccac
ccagaagaatggacggttgt
acagccggaagagtttctca
tggtcatatgcatgccaact
cccttcttcacgagcacttc
ctgcatgttcccaaaggatt
gactgggctgtgatctccat
cactaacggcaagagcatga
gattccctcaacaacccaga
tcatttggagtgaacgtgga
agcttgctgacgagatggtt
aatcctttgggaacatgcag
attagaatccaggcggcttt
glyceraldehyde 3-phosphatase
dehydrogenase
alpha-fetoprotein
albumin
DNA methyltransferase 1
gagtcaacggatttggtcgt
ttgattttggagggatctcg
agcttggtggtggatgaaac
ccagagatttcccaaagctg
gagctaccacgcagacatca
tctgcaatgacagcctcaag
tgggatttttccaacagagg
cgaggaagtagaagcggttg
Mouse
Gapdh
Afp
Alb
G6pc
Tat
Ck19
Dnmt1
Dnmt3a
Dnmt3b
c-Met
Cyp17a1
Cyp3a11
Cyp1a2
Cyp3a41
Cyp3a44
Human
GAPDH
AFP
ALB
DNMT1
All primers were designed using Primer3 v.0.4.0 (http://frodo.wi.mit.edu/primer3) and purchased
from Invitrogen.
10
Supporting Table 4. List of primers used for COBRA.
Primer name
Primer sequence
Primer position
hsa-miR-148a-B1
hsa-miR-148a-B2
TTTTTGTTGGGTATTTGTTTTTGTT
ACACTTAAACCCCCTCTAACCTTAC
S1701
AS2074
mmu-miR-148a-B1
mmu-miR-148a-B2
TTTTTTTTATTGGATTTTTATTTTT
CAAATTCTTCACAAAACCCTTATAC
S316
AS718
hALB-B3
hALB-B4
GGTAGTTAATGAAATATAAAGATGAGTTTA
AAAATTACCCACTTCATTATACCAAAA
S312
AS460
mAlb-B5
mAlb-B6
TGTTGTTTTATATAATTTTAATGAATGGAT
TTACCCACTTCATTTTACCAAAAAC
S56
AS344
11