Supplementary Material

Supplementary Materials and Methods
Raw miRNA microarray data analysis, miRNA qRT-PCR detection, and mRNA detection
The Affymetrix miRNA QC Tool was used for data summarization, normalization, and quality
control. SAM (significance analysis of microarrays) software was used to identify significantly
differentially expressed miRNAs. Only those with q-value less than 0.05 and expression increase
(or decrease) greater than 1.5-fold between two groups, were considered significantly changed.
Supervised
hierarchical clustering of
significantly
differentially
expressed
miRNAs
was built using Cluster 3.0 software and visualized with Java Treeview tools based on the array
spot median centered signal intensities of miRNA expression.
As we described previously [1], for miRNA detection, a polyA tail was added to the total RNA
using the E.coli polyA polymerase (NEB). 0.2 μg of the tailed total RNA was reverse transcribed
with ImProm-II (Promega). SYBR Green (Takara) qRT-PCR was performed to analyze miRNA
expression with the specific forward primers and the universal reverse primer complementary to
the anchor primer using the Applied Biosystems 7900HT Fast Real-Time PCR System. 18S
rRNA was used as reference gene. The mRNA was reverse transcribed with MMLV (Promega)
and quantified using SYBR Green (Takara) qRT-PCR. The primers that were used are listed in
supplementary Table S2.
Plasmids and lentivirus packaging
To construct the miRNA-expressing plasmids, the DNA fragment encoding pre-miRNA
(flanking upstream and downstream 30-50nt) was amplified using genomic DNA from the
human cell line HEK293T as the PCR template. The fragment was inserted into the expression
vector pSilencer4.1-CMV-puro (Ambion) and the lentiviral expression vector pCDH-EF1-MCST2A-Puro (Systems Biosciences). To construct the 3’UTR (3’ untranslated region) luciferase
reporter plasmids of the predicted target genes, the putative miRNA binding sites in the 3’UTR
of the target genes were selected according to the analysis with the online TargetScan software
(http://www.targetscan.org/index.html). The DNA fragments containing putative binding sites in
their 3’-UTRs were amplified using PCR and cloned into the XbaI site immediately downstream
of the stop codon in the pGL3-promoter vector (Promega). The primers that were used are listed
in supplementary Table S2.
Packaging of the pCDH-CMV-MCS-EF1-T2A-Puro based miRNA expression constructs (or
empty vector) into pseudoviral particles was performed using the pPACKH1™ HIV Lentivector
Packaging Kit according to the manufacturer's instructions (LV500A-1, Systems Biosciences).
The virus-containing supernatants were collected, filtered (with 0.45 µm syringe filters), and
used to infect the cultured P19 cells or primary embryonic mouse cardiomyocytes in complete
media containing 5 µg/ml polybrene (Sigma) after determining the titer of the lentiviral particles.
Cardiogenic differentiation of P19 cells
Differentiation was routinely induced with DMSO as Wobus previously described [2]. Briefly,
P19 cells were cultured in complete DMEM medium supplemented with 1% DMSO (Sigma) as
an inducer of cardiac differentiation. Drops of differentiation medium (50 μl) containing 1000
cells were placed onto the lids of Petri dishes filled with DMEM and cultured for 2 days. Cells in
the hanging drops were transferred into bacteriological grade Petri dishes to further develop into
embryoid bodies (EBs) for two more days. Approximately 50 EBs were collected and placed
onto 10-cm tissue culture grade dishes and grown for an additional 6 days before the analysis.
Luciferase assay
HEK293 cells were seeded in 96-well plates. The cells were cotransfected with 5 ng of the
internal control vector pRL-renilla (Promega), 50 ng of the different 3’UTR pGL3-promoter
reporters, and 150 ng of the miRNA expression plasmids (or pSilencer4.1 CMV-negative). At 48
hr after transfection, the firefly and renilla luciferase activities were assayed using the Dual-Glo
Luciferase Assay System (Promega). All experiments were performed in triplicate and repeated
at least three times.
Bioinformatic analysis of the miRNA target genes
Experimentally validated miRNA target genes were computationally screened with the
miRTarBase database (http://mirtarbase.mbc.nctu.edu.tw/index.html), and the predicted miRNAs
target
genes
were
screened
using
(http://www.targetscan.org/index.html),
several
PicTar
online
programs,
including
(http://pictar.bio.nyu.edu/),
and
TargetScan
miRanda
(http://microrna.sanger.ac.uk). The genes that were associated with heart development and
congenital heart disease were derived by searching the database of IPA (Ingenuity Pathways
Analysis). The visualized network of miRNAs and target genes was generated using IPA
Ingenuity Systems.
Reference:
[1] Zhang J, Du YY, Lin YF, Chen YT, Yang L, Wang HJ, et al. The cell growth suppressor, mir-126, targets IRS-1.
Biochemical and biophysical research communications. 2008;377:136-40.
[2] Wobus AM, Kleppisch T, Maltsev V, Hescheler J. Cardiomyocyte-like cells differentiated in vitro from embryonic
carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels. In vitro
cellular & developmental biology Animal. 1994;30A:425-34.
Supplementary Table S1. Information on RVOT myocardium tissues of TOF and normal RVOT tissues
TOF
Norm
al
tissue ID
gender
age (month)
Syndrome or
causes of death
T5 #
T7 #
T13 #
T16 #
T25 #
T29
T51
T54
T62
T76
T24
T28
T38
T39
T40
T41
T44
T46
T53
T55
T128
T129
T132
T8
T11
T114
T119
T3
T33
T74
T9
N1 #
N2 #
N3 #
N4
N5
N6
N7
N8
N9
M
F
F
M
M
M
M
F
M
M
M
M
M
F
F
M
M
M
M
F
M
F
M
M
M
F
M
M
M
F
F
F
F
M
M
F
F
F
F
M
35
27
25
32
24
10
8
5
12
5
39
24
12
36
12
7
5
8
10
12
2
8
12
12
14
12
8
5
10
8
8
12
18
2
24
2
37
5
27
32
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF/PDA
TOF
TOF
TOF
TOF
TOF
TOF
TOF
TOF/PDA
traffic accident
traffic accident
suffocation
traffic accident
suffocation
traffic accident
suffocation
traffic accident
traffic accident
TOF: Tetralogy of Fallot;
PDA: Patent ductus arteriosus
#: tissues used in microarray assays
Supplementary Table S2. Primer sequence
Primer name
Primer sequence
hsa-miR-130b RF
hsa-miR-187 RF
hsa-miR-154 RF
hsa-miR-363 RF
hsa-miR-21 RF
hsa-miR-222 RF
hsa-miR-19a RF
hsa-miR-708 RF
hsa-miR-382 RF
hsa-miR-181d RF
hsa-miR-887 RF
hsa-miR-424star RF
hsa-miR-192 RF
hsa-miR-155 RF
hsa-miR-503 RF
hsa-miR-214star RF
hsa-miR-93star RF
hsa-miR-886 3P RF
hsa-miR-29b2 star RF
hsa-miR-29c RF
hsa-miR-150 RF
hsa-miR-139 5p RF
hsa-miR-30a star RF
hsa-miR-499-3p RF
hsa-miR-146b
hsa-miR-181C RNF
hsa-miR-194 RNF
hsa-miR-494 RNF
18S polyA RNF
hsa-miR-424 RF
hsa-miR-660 RF
hsa-miR-148b RF
hsa-miR-140-5p RF
hsa-miR-346 RF
hsa-miR-381 RF
hsa-miR-345 RF
hsa-miR-330-3p RF
hsa-miR-148a RF
hsa-miR-132 RF
hsa-miR-337-5p RF
hsa-miR-181a-star RF
hsa-miR-720 RF
XBA1 HAS2 utrF
XBA1 HAS2 utrT
XBA1 NF1 utrF
XBA1 NF1 utrT
gtgccgCAGTGCAATGATGAAAG
gtgccgTCGTGTCTTGTGTTGCAG
acgtccTAGGTTATCCGTGTTGCCTTC
acgtccAATTGCACGGTATCCATC
gtgccgTAGCTTATCAGACTGATG
gtgccgAGCTACATCTGGCTACTG
gtgccgTGTGCAAATCTATGCAAAAC
gtgccgAAGGAGCTTACAATCTAGCTG
gtgccgGAAGTTGTTCGTGGTGGATTC
gtgcTgAACATTCATTGTTGTCGGTGG
gtgcTgGTGAACGGGCGCCATCCCGAG
gtgcTgCAAAACGTGAGGCGCTG
gtgcTgCTGACCTATGAATTGACAG
gtgcTgTTAATGCTAATCGTGATAG
gtgcTgTAGCAGCGGGAACAGTTCTG
gtgcTgTGCCTGTCTACACTTGCTG
gtgcTgACTGCTGAGCTAGCACTTC
gtgcTgCGCGGGTGCTTACTGAC
gtgcTgCTGGTTTCACATGGTGGCTTAG
gtgcTgTAGCACCATTTGAAATCGGT
gtgcTgTCTCCCAACCCTTGTACCAG
gtgcTgTCTACAGTGCACGTGTCTC
gcTgCTTTCAGTCGGATGTTTGCAG
gtgcTgAACATCACAGCAAGTCTG
gtgccgTGAGAACTGAATTCCA
gtgtgggAACATTCAACCTGTCGGTG
gtgccgTGTAACAGCAACTCCA
gtgcTgTGAAACATACACGGGAAAC
agtcgtaacaaggtttccgtaggtg
gtggtCAGCAGCAATTCATGTTTTG
gtgcTgTACCCATTGCATATCGGAG
gtgcTgTCAGTGCATCACAGAAC
gtgcTgCAGTGGTTTTACCCTATG
gtgcTg TGTCTGCCCGCATGCCTGCCTC
gtgcTg TATACAAGGGCAAGCTCTC
gtgcTg GCTGACTCCTAGTCCAG
gtgcTg GCAAAGCACACGGCCTGCAG
gtgcTg TCAGTGCACTACAGAACTTTG
gtgcTg TAACAGTCTACAGCCATG
gtgcTg GAACGGCTTCATACAGGAG
gtgcTg ACCATCGACCGTTGATTG
gtgcTg TCTCGCTGGGGCCTC
GCG TCTAGAttgacgtttgcagtcacaca
GCG TCTAGA aaaacactttcaggcggatg
GCG TCTAGA ggaaaataagtgcgaccaca
GCG TCTAGA tgaaatcctctaatgaaacagcac
MiR-424 preF
MiR-424 preT
miR-222 preF
miR-222 preT
ggcttccttcagtcatccag
gcacctggtggcaggaacac
tcccttccagaatctctcttca
tgcctatgtgtgcatgtgtg
GATA4 RF
GATA3 RT
NKX2.5 RF
NKX2.5 RT
TBX5 RF
TBX5 RT
αMHC RF
αMHC RT
NF1 RF
NF1 RT
HAS2 RF
HAS2 RT
has-miR-1 RF
has-miR-27a/b RF
has-miR-133a RF
actctggaggcgagatggg
ctcggcattacgacgccacag
TCTCCGATCCATCCCACTTTATTG
TTGCGTTACGCACTCACTTTAATG
CACAGCCCCTTCAGCAGCGAGAC
AGGGGCCCCGAGGTGAAATGAG
GAGGACCAGGCCAATGAGTA
GCTGGGTGTAGGAGAGCTTG
taactttgcattggttggacac
ccacgctctgtgtattcacttc
gtcatgtacacagccttcagag
actgctgaggaatgagatccag
gtatgTGGAATGTAAAGAAGTATG
gttcTTCACAGTGGCTAAGTTC
ccagTTGGTCCCCTTCAACCAG
Supplementary Table S3. Aberrant miRNA expression in TOF heart tissues by microarray
miRNA
name
miR-130b
miR-132
miR-139-5p
miR-140-5p
miR-146b-5p
miR-148a
miR-148b
miR-152
miR-154
miR-155
miR-181a*
miR-181c
miR-181d
miR-187
miR-192
miR-194
miR-19a
miR-21
miR-214*
miR-222
miR-29b-2*
miR-29c
miR-30a*
miR-330-3p
miR-337-5p
miR-345
miR-346
miR-363
miR-381
miR-382
miR-424
miR-424*
miR-494
miR-499-3p
miR-503
miR-660
miR-708
miR-720
miR-886-3p
miR-887
miR-93*
average expression
TOF
normal
123.70
45.24
163.02
79.20
226.21
550.60
86.92
35.56
73.67
17.02
34.29
16.57
43.50
17.35
274.19
714.34
36.58
17.03
148.07
62.39
38.62
81.33
90.47
48.47
92.37
49.85
43.43
21.28
45.41
22.27
86.49
46.92
38.80
18.71
111.57
50.64
40.32
16.27
3071.05 1435.62
62.23
190.39
61.14
170.99
148.83
321.10
37.01
16.38
48.72
20.57
179.87
79.18
40.87
17.53
69.27
34.05
37.70
16.26
50.35
21.68
24.79
14.13
60.36
26.29
462.30
1012.03
37.79
17.50
94.87
34.67
60.72
18.26
74.04
32.25
314.39
925.95
96.86
46.99
69.69
32.28
43.31
16.96
Fold change
(TOF/normal)
2.73
2.06
0.41
2.44
4.33
2.07
2.51
0.38
2.15
2.37
0.47
1.87
1.85
2.04
2.04
1.84
2.07
2.20
2.48
2.14
0.33
0.36
0.46
2.26
2.37
2.27
2.33
2.03
2.32
2.32
1.76
2.30
0.46
2.16
2.74
3.33
2.30
0.34
2.06
2.16
2.55
q-value
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.05
0.01
0.00
0.02
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.02
0.00
0.01
0.00
0.00
0.01
0.00
0.00
0.00
0.05
0.01
0.00
0.00
Supplementary Table S4. miRNAs with no significant changes by real-time RT-PCR quantification
miRNA
name
miR-1 #
miR-132
miR-133 #
miR-139-5p
miR-140-5p
miR-148a
miR-148b
miR-152
miR-187
miR-194
miR-21
miR-214*
miR-27 #
miR-29b2*
miR-30a*
miR-330-3p
miR-345
miR-346
miR-381
miR-382
miR-494
miR-499-3p
miR-503
miR-886-3p
miR-887
miR-93*
Average
TOF
32.5801
0.0139
48.4642
0.0426
0.0378
0.0357
0.0477
0.0198
0.9123
0.9080
63.5907
0.4288
3.4113
0.0170
0.4201
0.1097
1.2335
0.0742
0.2141
0.0126
13.6356
0.0570
0.0204
1.1919
0.1739
0.0059
expression
normal
38.0472
0.0128
32.5049
0.0294
0.0363
0.0178
0.0253
0.0097
0.2126
0.5192
20.8590
0.2656
2.1668
0.0221
0.3917
0.0553
0.3928
0.0617
0.2023
0.0070
13.3686
0.0263
0.0120
1.1846
0.0460
0.0023
Fold change
(TOF/normal)
0.8563
1.0898
1.4910
1.4505
1.0414
2.0083
1.8836
2.0348
4.2917
1.7489
3.0486
1.6143
1.5743
0.7710
1.0726
1.9858
3.1402
1.2042
1.0584
1.7983
1.0200
2.1628
1.6941
1.0062
3.7826
2.5392
p-Value
0.3279
0.0631
0.5787
0.7173
0.9807
0.9039
0.7537
0.0865
0.9423
0.0865
0.1688
0.9807
0.6783
0.4033
0.9807
0.0865
0.8327
0.9423
0.2369
0.4257
0.2735
0.0505
0.4257
0.3927
0.1688
0.2993
#: miRNAs which were previously described to be involved in heart development, but not identified in our
microarray assay.
Supplementary Table S5. Genes experimentally validated or predicted to be targeted by the identified
miRNAs
miRNAs
Target genes
miR-146b-5p
DTNA, NOTCH1, PBX2, RARB, ROR1, EGFR, PCSK6, RHOA, NOTCH1, TGIF1
miR-155
ACVR2A, ACVR2B, CHD7, CTNNB1, CYR61, DSG2, MEIS1, NR2F2,
PCSK6, PLXND1, RHOA, SALL1, SOX11
miR-19a
ADRB1, BMPR2, DTNA, ERBB3, FOXP1, FRS2, FURIN, GJA1, HEG1, HHEX,
ID2, KIF3A, LUZP1, MAPK14, MED13L, MKL2, NF1, NFATC1, PTEN, SIN3B,
SOX12, SOX4, TEK, TGFBR3, TGIF1
miR-222
ADRA2B, CRKL, FOXP1, GATA4, NRG1, PTEN, SH3PXD2B, TFAP2A, ZFPM2,
miR-363
BMP4, HAND1, HAND2, MTHFR, NF1, NKX2-5, PAX3, PCSK6, SMAD7
ACVR1, EDN1, ERBB3, FOXP1, GJA1, HEG1, HES1, HHEX, PSEN1, RYR2,
SOX4, TEK, TGFB2, ZFPM2
miR-130b
miR-660
CHD7, DTNA, KIF3A, MEIS1, RFX3
miR-424/424*
CCNE1, FGF8, GPC3, HAS2, HEY2, JUP, NF1, NRP1, PAX3, PCSK6, TBX1
miR-154
PTEN
miR-181c/d
ACVR2A, ACVR2B, ADRBK1, BMPR2, CHD7, CYR61, DTNA, FOXP1,
FRS2, GATA6, HAND2, HEY2, KIF3A, LEFTY2, LUZP1, MYH10, NOTCH2,
NRP1, PBX1, PKP2, PROX1, PTEN, SEMA3C, SIN3B, SMAD7, SRSF5,
TGFBR3, TLL1, VCL
miR-708
CRELD1, FOXP1, NTF3, PITX2
miR-192
ACVR2A, ACVR2B
miR-29c
ACVR2A, DSC2, FRS2, HAND1, HEY2, KIF3A, LUZP1, NOTCH2, PDGFC,
PSEN1, RARB, SH3PXD2B, SOX12, TGFB2, TLL1, VCL
miR-181a*
FBLN1, PAX3, PCSK6
Underlined target genes had been experimentally validated.
Supplementary Figure S6. Flow diagram of the design

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