Supplementary Material.
Mehta et al., Gene and miRNA Transcriptional Signatures of Angiotensin II in Endothelial Cells.
Methods
Microarray and pathway analysis
Total RNA samples were purified using the RNease Kit (Qiagen) and quantified with a Ribogreen fluorescence
assay on a Biotek Synergy 4 Plate Reader. Quality of total RNA was assessed using an Agilent Total RNA
6000 NanoKit on the Agilent 2100 Bioanalyzer. The Illumina TotalPrep RNA amplification kit was used to
prepare biotinylated antisense RNA by starting with 500ng total RNA, incubating 14 hours at 37°C, and elution
from a column into 100μl of nuclease-free H2O. Quality and quantity of the cRNA was determined by
Ribogreen fluorescence and Agilent Bioanalyzer electropherograms. Then, 750 ng of cRNA (in a total volume
of 5μl) was loaded onto the Illumina Human HT-12 v4 Gene Expression BeadChip for hybridization (17 hours
at 58°C). After blocking, staining, and washing of the microarrays, they were scanned on the Illumina iScan;
data was imported into GenomeStudio v2010.3 for analysis by the Gene Expression v1.8.0 module. Internal
microarray quality control data was assessed for performance of BeadChip and experiment; a purchased RNA
quality control was included in each step of the RNA preparation, quantitation, and hybridization to
microarrays. Sample quality was determined using scatterplots and hierarchical clustering. Prior to group or
differential gene expression analyses, raw microarray data were subjected to background subtraction and
average normalization on GenomeStudio. For differential gene expression, the Illumina Custom Model was
selected. This model assumes that target signal intensity (I) is normally distributed among replicates. The
variation has three components: biological variation (σbio), nonspecific biological variation (σneg), and technical
error (σtech). Standard deviation exceeding σtech is assumed to reflect biological variation. For selection of
differentially expressed genes, we utilized p < 0.05 cut-off value. Gene annotation and pathway analysis was
performed using the Expression Analysis Systematic Explorer (EASE, 11).
RNA Quality Assessment and Quantification:
Total RNA quantification was determined by UV/Visible absorbance (NanoDrop 8000, ThermoFisher
Scientific, Wilmington, DE) according to the manufacturer's instructions.
RNA quality and small RNA
extraction efficiency was evaluated using the Small RNA Kit with the Agilent 2100 Bioanalyzer (Agilent
Technologies, Wilmington, DE). Absolute miRNA content (pg/µL) and its percentage relative to small RNA
content (ng/µL) was calculated (% miRNA/small RNA ratio).
Reverse Transcription and Quantitative Real-Time PCR:
The starting material for mature miRNA expression profiling was 500 ng of total RNA per sample,
containing mature miRNAs, small nucleolar and small nuclear RNAs. Reverse transcription using the miScript
II RT kit (Qiagen, SA Biosciences, Valencia, CA) per the manufacturer's directions selectively converted
mature miRNAs into cDNA. The cDNA synthesis product was mixed with miScript SYBR Green PCR Kit,
miScript Universal Primer and QuantiTect SYBR Green PCR Master mix (Qiagen, SA Biosciences, Valencia,
CA) using a set protocol. Real-time PCR profiling of the 384HC miScript miRNA PCR array (Qiagen, SA
Biosciences, Valencia, CA) was performed on an ABI7900HT Fast Real-Time PCR System (Life Technologies,
Grand Island, NY). Cycling conditions for real-time PCR included the following steps: activation step (15 min
at 95°C) followed by 40 cycles of denaturing (15 sec at 94°C), annealing (30 sec at 55°C), and extension (30
sec at 70°C). A dissociation curve analysis of the PCR product consisted of a 2-step cycling program (15 sec at
94°C and 15 sec at 60°C) after the 3-step cycling process. The baseline was set from cycle 3 to cycle 15 and the
manual threshold was defined as 0.10 for all samples. CT scores were exported from the real-time PCR
instrument and uploaded into the RT2 Profiler PCR Array Data Analysis v3.5 software (Qiagen, SA
Biosciences, Valencia, CA) for preliminary data analysis using the ΔΔCT method of relative quantitation.
Quantitative PCR
RNA was purified using the miRNAeasy kit (Qiagen, Valencia, CA), and cDNA was synthesized using
the iScript cDNA synthesis kit (Bio-Rad) with 0.5 μg of total RNA according to the manufacturer's
recommendations. qPCR was performed with pre-designed primers selected from PrimerBank and ordered from
Integrated DNA Technologies (Coralville, IA). RT qPCR was performed using the Applied Biosystems 7900
real-time PCR system. All qPCR reactions were carried out in a final volume of 15 μl containing 1X of SYBR
Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), 300 nmol/L of each gene specific primers, 100 ng
cDNA, in sterile deionized water. The standard cycling condition was 50°C for 2 min, 90°C for 10 min,
followed by 40 cycles of 95°C for 15 s and 62°C for 1 min. The results were analyzed using SDS 2.3 relative
quantification manager software. The comparative threshold cycles values were normalized for glyceraldehyde
3-phosphate dehydrogenase (GAPDH) reference genes. qPCR was performed in triplicate to ensure quantitative
accuracy.
Supplemental Table 1. Genes differentially expressed (>1.5-fold, p<0.05) following Ang II exposure (10-8M)
SYMBOL
CLDN23
PPP4R4
GPR137C
ID2
ATOH8
RASL10A
SMAD6
LOC202227
PTGS2
MYCN
HEY2
ADAMTS1
PCDH17
CENTA1
LOC100132394
KITLG
CXXC5
SMAD9
LOC100134364
CHSY3
LOC728510
NEDD9
C8orf4
TRAPPC2P1
LOC100133565
DDIT4L
FAM46A
LOC100008589
HLX
FILIP1L
LOC441763
MEF2C
ARID5B
RCAN1
PPP1R3C
SERPINB2
ELL2
GOLPH3
SOX17
OSGIN2
KRT10
DUSP23
Ang II Ang II Diff
2 hrs
12 hrs Pval*
0.91
0.01
0.0015
1.00
0.03
0.0050
0.79
0.04
0.0000
0.88
0.07
0.0088
1.13
0.08
0.0002
0.87
0.08
0.0055
1.05
0.12
0.0000
1.08
0.12
0.0005
0.96
0.14
0.0000
0.92
0.14
0.0000
1.01
0.17
0.0000
0.94
0.17
0.0000
0.95
0.23
0.0000
1.20
0.23
0.0154
0.97
0.25
0.0167
0.99
0.27
0.0004
1.02
0.28
0.0000
1.17
0.30
0.0244
1.13
0.31
0.0000
1.04
0.32
0.0000
0.89
0.34
0.0025
0.93
0.36
0.0206
1.18
0.36
0.0016
0.93
0.36
0.0388
1.07
0.37
0.0000
1.02
0.37
0.0003
0.90
0.38
0.0001
0.95
0.38
0.0009
1.03
0.39
0.0016
0.91
0.40
0.0105
1.12
0.40
0.0000
1.06
0.47
0.0064
0.91
0.47
0.0140
1.05
0.48
0.0062
0.95
0.49
0.0390
1.05
0.49
0.0147
0.80
0.50
0.0354
1.17
0.51
0.0285
0.94
0.52
0.0123
1.07
0.52
0.0406
0.93
0.53
0.0101
0.90
0.53
0.0089
DEFINITION
claudin 23
protein phosphatase 4, regulatory subunit 4
G protein-coupled receptor 137C
dominant negative helix-loop-helix protein
atonal homolog 8 (Drosophila)
RAS-like, family 10, member A, transcript variant 2
SMAD family member 6, transcript variant 1,
PREDICTED: misc_RNA, miscRNA.
prostaglandin-endoperoxide synthase 2
v-myc myelocytomatosis viral related oncogene
hairy/enhancer-of-split related with YRPW motif 2
ADAM metallopeptidase with thrombospondin type 1 motif
protocadherin 17
centaurin, alpha 1
PREDICTED: hypothetical protein
KIT ligand, transcript variant b
CXXC finger 5
SMAD family member 9
PREDICTED: hypothetical protein
chondroitin sulfate synthase 3 (CHSY3),
PREDICTED: hypothetical LOC728510
neural precursor expressed, developmentally down-regulated 9
chromosome 8 open reading frame 4
trafficking protein particle complex 2 pseudogene 1
PREDICTED: similar to hCG23738
DNA-damage-inducible transcript 4-like
family with sequence similarity 46, member A
28S ribosomal RNA, non-coding RNA
H2.0-like homeobox
filamin A interacting protein 1-like, transcript variant 2
PREDICTED: hypothetical LOC441763
myocyte enhancer factor 2C
AT rich interactive domain 5B (MRF1-like)
regulator of calcineurin 1, transcript variant 3,
protein phosphatase 1, regulatory (inhibitor) subunit 3C
serpin peptidase inhibitor, clade B (ovalbumin), member 2
elongation factor, RNA polymerase II
golgi phosphoprotein 3 (coat-protein)
SRY (sex determining region Y)-box 17
oxidative stress induced growth inhibitor family member 2
keratin 10
dual specificity phosphatase 23
CKS2
RGS4
LOC646753
TMEM14D
MED30
CENPN
CCDC59
TNFAIP8L3
SOX18
C21orf55
KAT2A
LOC100134159
KLHL28
NOTCH1
LOC100133607
LOC400721
ANPEP
LOC100132564
LAMA4
NAGLU
CARD8
LOC100133233
ROBO4
AXUD1
LOC728888
DOCK6
SHROOM2
PGF
KLHL3
IL18
RHBDF1
MBD4
LOC100131989
DENND4B
LOC440704
PAFAH1B3
PNPT1
STAB1
PLD1
GDF15
DDIT4
FLJ25363
LAMB2
D2HGDH
CCDC130
LOC399900
WSB1
1.13
0.96
0.95
1.03
0.86
0.93
1.02
0.96
0.94
0.91
0.99
0.90
0.85
0.84
1.09
0.97
0.91
1.04
0.98
0.96
0.94
1.07
1.09
1.22
0.95
1.19
0.90
1.28
0.75
1.13
0.84
0.77
0.81
1.16
1.08
1.15
0.96
0.93
0.95
1.19
0.97
0.93
0.90
0.92
1.01
0.98
0.94
0.55
0.55
0.56
0.57
0.57
0.58
0.58
0.59
1.89
1.90
1.93
1.93
1.93
1.95
1.95
1.96
1.97
1.98
1.98
2.00
2.01
2.05
2.06
2.07
2.07
2.07
2.09
2.10
2.10
2.11
2.12
2.12
2.12
2.14
2.15
2.15
2.16
2.16
2.16
2.16
2.16
2.18
2.19
2.20
2.20
2.21
2.22
0.0090
0.0190
0.0254
0.0244
0.0366
0.0424
0.0370
0.0473
0.0436
0.0423
0.0446
0.0363
0.0352
0.0328
0.0311
0.0268
0.0286
0.0359
0.0331
0.0442
0.0286
0.0161
0.0464
0.0248
0.0190
0.0182
0.0333
0.0173
0.0279
0.0175
0.0115
0.0161
0.0416
0.0430
0.0469
0.0287
0.0358
0.0291
0.0129
0.0149
0.0189
0.0243
0.0134
0.0340
0.0085
0.0083
0.0036
CDC28 protein kinase regulatory subunit 2
regulator of G-protein signalling 4
PREDICTED: similar to ribosomal protein S26
PREDICTED: transmembrane protein 14D
mediator complex subunit 30
centromere protein N
coiled-coil domain containing 59
tumor necrosis factor, alpha-induced protein 8-like 3
SRY (sex determining region Y)-box 18
chromosome 21 open reading frame 55
K(lysine) acetyltransferase 2A
PREDICTED: similar to Coiled-coil domain containing 144B
kelch-like 28 (Drosophila)
Notch homolog 1, translocation-associated (Drosophila)
hypothetical protein LOC100133607, transcript variant 2
PREDICTED: similar to Zinc finger protein 418
alanyl (membrane) aminopeptidase (aminopeptidase N
PREDICTED: hypothetical protein LOC100132564
laminin, alpha 4
N-acetylglucosaminidase, alpha
caspase recruitment domain family, member 8
PREDICTED: hypothetical protein LOC100133233
roundabout homolog 4, magic roundabout (Drosophila)
AXIN1 up-regulated 1
PREDICTED: similar to Protein KIAA0220
dedicator of cytokinesis 6
shroom family member 2
placental growth factor
kelch-like 3 (Drosophila)
interleukin 18 (interferon-gamma-inducing factor)
rhomboid 5 homolog 1 (Drosophila)
methyl-CpG binding domain protein 4
PREDICTED: hypothetical protein LOC100131989
DENN/MADD domain containing 4B
PREDICTED: hypothetical gene supported by BC042042
platelet-activating factor acetylhydrolase, isoform Ib, gamma
polyribonucleotide nucleotidyltransferase 1
stabilin 1
phospholipase D1, phosphatidylcholine-specific
growth differentiation factor 15
DNA-damage-inducible transcript 4
PREDICTED: similar to hypothetical protein FLJ25976
laminin, beta 2 (laminin S)
D-2-hydroxyglutarate dehydrogenase
coiled-coil domain containing 130
hypothetical gene supported by AK093779
WD repeat and SOCS box-containing 1, transcript variant 2
HIATL2
CLK2
LOC654103
PCDH12
SLC25A37
NBEAL2
LOC648921
NDST1
PIP5K2B
CEP27
FLJ35390
FNBP4
ADM
MAN2C1
LOC100133772
HCG2P7
TMEM191A
MIR1978
LOC440348
LOC100132727
SPSB1
MORC2
LOC653778
LOC729603
NOL12
LOC728457
LOC100130835
C15orf52
C8orf45
HS.224794
BHLHB2
LOC88523
EID2B
C1orf63
ETS2
XIST
STAG3L2
N4BP2
RAXL1
NUBPL
ADCY4
FAM156B
GBA2
FLJ46309
RBM33
CHST15
ZNF69
0.95
1.15
1.01
0.83
1.00
0.99
0.74
1.16
0.94
0.89
1.04
1.08
1.07
0.72
1.00
0.97
0.90
0.85
1.09
0.95
0.89
0.98
1.17
0.89
0.92
0.92
1.03
0.90
0.83
1.15
0.93
0.82
1.03
0.96
0.92
1.11
0.97
1.06
0.91
0.88
1.09
1.21
0.99
1.07
0.72
1.03
1.00
2.23
2.24
2.27
2.27
2.29
2.29
2.30
2.31
2.32
2.32
2.32
2.34
2.35
2.38
2.39
2.39
2.40
2.41
2.41
2.44
2.45
2.45
2.46
2.46
2.46
2.47
2.50
2.50
2.50
2.50
2.52
2.53
2.53
2.53
2.53
2.53
2.55
2.55
2.55
2.55
2.56
2.56
2.58
2.58
2.59
2.60
2.61
0.0073
0.0498
0.0093
0.0268
0.0158
0.0165
0.0333
0.0386
0.0045
0.0140
0.0100
0.0056
0.0031
0.0176
0.0193
0.0477
0.0310
0.0048
0.0226
0.0096
0.0089
0.0123
0.0047
0.0380
0.0092
0.0299
0.0026
0.0017
0.0401
0.0363
0.0014
0.0121
0.0369
0.0208
0.0067
0.0028
0.0137
0.0352
0.0089
0.0046
0.0016
0.0054
0.0448
0.0303
0.0207
0.0019
0.0206
hippocampus abundant transcript-like 2
PREDICTED: CDC-like kinase 2, transcript variant 3
PREDICTED: similar to solute carrier family 25, member 37
protocadherin 12
solute carrier family 25, member 37
neurobeachin-like 2
PREDICTED: similar to LOC283693 protein
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
phosphatidylinositol-4-phosphate 5-kinase, type II, beta
centrosomal protein 27kDa
hypothetical LOC255031 (FLJ35390), transcript variant 1
formin binding protein 4
adrenomedullin
mannosidase, alpha, class 2C, member 1
PREDICTED: similar to MCT
HLA complex group 2 pseudogene 7, non-coding RNA.
PREDICTED: misc_RNA, miscRNA.
microRNA 1978, microRNA.
similar to nuclear pore complex interacting protein
PREDICTED: hypothetical protein LOC100132727
splA/ryanodine receptor domain and SOCS box containing 1
MORC family CW-type zinc finger 2
PREDICTED: similar to solute carrier family 25, member 37
calcium binding protein P22 pseudogene, non-coding RNA
nucleolar protein 12
PREDICTED: misc_RNA (LOC728457), miscRNA.
PREDICTED: misc_RNA (LOC100130835), miscRNA.
chromosome 15 open reading frame 52
chromosome 8 open reading frame 45
cDNA FLJ33375 fis, clone BRACE2006137
basic helix-loop-helix domain containing, class B, 2
CG016 (LOC88523),
EP300 interacting inhibitor of differentiation 2B
chromosome 1 open reading frame 63, transcript variant 1,
v-ets erythroblastosis virus E26 oncogene homolog 2 (avian)
X (inactive)-specific transcript (non-protein coding)
stromal antigen 3-like 2
Nedd4 binding protein 2
retina and anterior neural fold homeobox like 1
nucleotide binding protein-like
adenylate cyclase 4
family with sequence similarity 156, member B
glucosidase, beta (bile acid) 2
hypothetical protein LOC649598
RNA binding motif protein 33 (RBM33), transcript variant 1
carbohydrate sulfotransferase 15
zinc finger protein 69
LOC729090
IL10
SDHAP2
C5orf28
TDRD1
C14orf153
CHST1
ADAMTS9
C14orf82
ANO2
ZNF738
C9orf80
ALPP
FLJ44124
QRFPR
LOC100133298
LOC100128084
LOC613037
LRAP
FAM63A
USP49
CXCR4
C12orf51
LOC100132391
LOC100128288
HS.542993
LOC100129362
NOTCH4
HS.145444
LOC729120
MST1
HSPC268
RBM5
SLC4A11
LOC647389
LOC100129502
LOC100133516
CYTH2
CDKN2AIPNL
CHRNA5
LOC100130053
LOC100130168
XRCC2
LOC645452
MAGT1
CCBE1
C14orf85
1.02
0.92
0.87
1.38
0.91
0.90
1.07
0.88
0.97
1.18
0.96
0.98
1.02
1.04
0.89
0.91
0.90
0.98
1.02
0.94
1.03
1.10
1.24
0.98
0.99
0.84
0.98
0.98
0.53
0.95
1.15
0.97
0.90
1.01
0.95
1.02
0.84
0.94
0.97
0.94
0.97
0.93
1.05
0.84
0.85
1.11
1.11
2.61
2.62
2.62
2.63
2.64
2.64
2.65
2.65
2.66
2.67
2.68
2.69
2.71
2.71
2.71
2.71
2.72
2.73
2.74
2.74
2.74
2.75
2.76
2.76
2.78
2.78
2.80
2.81
2.81
2.82
2.82
2.83
2.83
2.84
2.84
2.84
2.84
2.84
2.85
2.92
2.92
2.93
2.94
2.95
2.96
2.97
2.99
0.0266
0.0285
0.0211
0.0281
0.0128
0.0271
0.0026
0.0021
0.0080
0.0370
0.0010
0.0273
0.0006
0.0005
0.0006
0.0165
0.0009
0.0485
0.0004
0.0387
0.0007
0.0475
0.0370
0.0032
0.0006
0.0010
0.0004
0.0007
0.0203
0.0030
0.0500
0.0005
0.0004
0.0031
0.0151
0.0010
0.0011
0.0046
0.0166
0.0122
0.0005
0.0002
0.0172
0.0003
0.0003
0.0168
0.0003
Similar to Eukaryotic translation elongation factor 1 alpha 1
interleukin 10
succinate dehydrogenase complex, subunit A, non-coding RNA
chromosome 5 open reading frame 28
tudor domain containing 1
chromosome 14 open reading frame 153
ADAM metallopeptidase with thrombospondin type 1 motif 9
PREDICTED: chromosome 14 open reading frame 82
anoctamin 2
PREDICTED: misc_RNA (ZNF738), partial miscRNA
chromosome 9 open reading frame 80
alkaline phosphatase, placental (Regan isozyme)
hypothetical protein LOC641737
pyroglutamylated RFamide peptide receptor
PREDICTED: hypothetical protein LOC100133298
PREDICTED: hypothetical protein LOC100128084
nuclear pore complex interacting protein pseudogene
leukocyte-derived arginine aminopeptidase
family with sequence similarity 63, member A
ubiquitin specific peptidase 49
chemokine (C-X-C motif) receptor 4, transcript variant 1
chromosome 12 open reading frame 51
PREDICTED: hypothetical protein LOC100132391
hypothetical protein LOC100128288, non-coding RNA
xj89b12.x1 Soares_NFL_T_GBC_S1 cDNA clone
PREDICTED: hypothetical protein LOC100129362
Notch homolog 4 (Drosophila)
cDNA FLJ11494 fis, clone HEMBA1001942
PREDICTED: hypothetical LOC729120
macrophage stimulating 1 (hepatocyte growth factor-like)
hypothetical protein HSPC268
RNA binding motif protein 5
solute carrier family 4, sodium borate transporter, member 11
PREDICTED: hypothetical protein LOC647389
PREDICTED: hypothetical protein LOC100129502
PREDICTED: hypothetical protein LOC100133516
cytohesin 2, transcript variant 1
CDKN2A interacting protein N-terminal like
cholinergic receptor, nicotinic, alpha 5
PREDICTED: hypothetical protein LOC100130053
PREDICTED: hypothetical protein LOC100130168
X-ray repair complementing defective repair in CHO cells 2
PREDICTED: similar to hCG1782414
magnesium transporter 1
collagen and calcium binding EGF domains 1
chromosome 14 open reading frame 85 non-coding RNA
ZNF14
ZNF786
C6orf170
HS.554507
LIME1
LOC401098
LOC255167
LRRC37B2
PRO1853
TNFRSF25
ZNF486
MEG3
RHBDL2
LOC100128126
BMS1P5
FAM73A
TMEM137
LOC729978
BLZF1
CATSPER2
FAM175A
HS.572444
LOC649841
LOC100190938
ZNF682
HS.544637
TMEM17
LOC728809
SEMA3E
SNORD31
CCDC125
SNORD36C
DTWD2
LOC730313
LOC100134868
SEMA4C
LOC90586
LOC100128098
LOC728105
KIFC2
RNY3
FAM153B
NLRP8
CTGLF3
LOC728903
FLJ40722
EFNA1
0.93
0.90
0.74
0.66
0.93
0.79
0.95
0.90
1.32
0.87
1.09
0.93
0.91
0.62
1.24
1.12
0.92
0.97
1.00
0.95
1.21
0.83
0.66
1.04
0.82
0.79
1.00
1.03
1.13
0.91
1.08
0.98
0.90
0.98
1.04
1.13
1.04
0.79
0.83
1.14
0.80
1.15
0.96
1.05
1.05
0.91
1.08
3.01
3.04
3.05
3.07
3.07
3.08
3.09
3.10
3.14
3.14
3.14
3.18
3.19
3.19
3.20
3.22
3.25
3.25
3.25
3.25
3.27
3.28
3.28
3.29
3.30
3.30
3.31
3.31
3.34
3.36
3.39
3.41
3.42
3.45
3.46
3.46
3.48
3.49
3.52
3.54
3.54
3.55
3.59
3.61
3.64
3.65
3.73
0.0122
0.0011
0.0401
0.0242
0.0084
0.0002
0.0176
0.0001
0.0021
0.0176
0.0002
0.0024
0.0372
0.0311
0.0001
0.0115
0.0340
0.0001
0.0001
0.0088
0.0002
0.0011
0.0044
0.0081
0.0381
0.0144
0.0115
0.0001
0.0123
0.0105
0.0205
0.0189
0.0074
0.0000
0.0167
0.0225
0.0074
0.0119
0.0087
0.0066
0.0018
0.0271
0.0115
0.0006
0.0074
0.0102
0.0047
zinc finger protein 14
zinc finger protein 786
chromosome 6 open reading frame 170
primary neuroblastoma cDNA, clone:Nbla10527
Lck interacting transmembrane adaptor 1
PREDICTED: misc_RNA (LOC401098), miscRNA
hypothetical LOC255167 (LOC255167), non-coding RNA
leucine rich repeat containing 37, member B2, non-coding RNA
hypothetical protein PRO1853, transcript variant 1
tumor necrosis factor receptor superfamily, member 25
PREDICTED: zinc finger protein 486
maternally expressed 3 (non-protein coding), transcript variant 1
rhomboid, veinlet-like 2 (Drosophila)
PREDICTED: hypothetical protein LOC100128126
BMS1 pseudogene 5, non-coding RNA
family with sequence similarity 73, member A
PREDICTED: transmembrane protein 137, misc RNA
PREDICTED: similar to LOC339047 protein, transcript variant 2
basic leucine zipper nuclear factor 1
cation channel, sperm associated 2, transcript variant 4,
family with sequence similarity 175, member A
cDNA: FLJ21679 fis, clone COL09221
Similar to protein immuno-reactive with anti-PTH antibodies
hypothetical LOC100190938, transcript variant 2, non-coding
zinc finger protein 682, transcript variant 1
xr14b10.x1 NCI_CGAP_Lu28 cDNA clone
transmembrane protein 17
PREDICTED: hypothetical LOC728809 (LOC728809)
sema domain, secreted, (semaphorin) 3E
small nucleolar RNA, C/D box 31
coiled-coil domain containing 125
small nucleolar RNA, C/D box 36C
DTW domain containing 2
PREDICTED: hypothetical LOC730313
hypothetical LOC100134868 (LOC100134868), non-coding RNA
sema domain, (semaphorin) 4C
PREDICTED: AOC3 pseudogene (LOC90586), misc RNA.
PREDICTED: hypothetical protein LOC100128098
PREDICTED: misc_RNA (LOC728105), miscRNA
kinesin family member C2
RNA, Ro-associated Y3 (RNY3), small cytoplasmic RNA.
family with sequence similarity 153, member B (FAM153B)
NLR family, pyrin domain containing 8
centaurin, gamma-like family, member 3
PREDICTED: hypothetical LOC728903
PREDICTED: hypothetical protein FLJ40722, transcript variant 4
ephrin-A1, transcript variant 1
SNORA24
LOC100128510
SNORA33
DUSP19
STAG3L3
1.10
1.12
0.98
0.91
1.01
3.74
3.76
3.76
3.82
3.89
HS.582113
LOC730995
LOC100130276
LOC100132585
SCARNA9
0.73
1.18
1.04
1.16
1.23
3.90
3.99
4.06
4.07
4.11
LOC132241
LOC100129211
LMOD3
MIR21
LOC791120
HS.535028
LOC389765
GOLGA8A
CLOCK
LOC648852
HES1
LOC653829
SNORD4A
LOC400304
SSTR2
AMT
SPTBN5
SNORA32
KIT
CYP26B1
LOC653158
FLJ44342
N4BP3
PABPC1L
1.05
4.14
0.84
4.21
1.34
4.31
0.97
4.32
0.83
4.36
0.82
4.47
1.19
4.52
0.89
4.60
0.95
4.64
0.97
4.92
1.26
5.04
0.92
5.17
0.96
5.26
1.51
5.28
1.25
5.57
1.03
5.59
0.90
5.60
1.01
5.71
1.06
6.38
2.44
6.58
0.77
7.77
1.29
9.94
1.08 12.11
2.40 15.42
1.00 63.13
24.89 170.60
0.0019 small nucleolar RNA, H/ACA box 24
0.0032 PREDICTED: hypothetical protein LOC100128510
0.0463 small nucleolar RNA, H/ACA box 33
0.0424 dual specificity phosphatase 19
0.0002 stromal antigen 3-like 3
SM011178 Brain 3 EST cDNA clone ID_11178 3', mRNA
0.0089 sequence
0.0216 PREDICTED: similar to ankyrin repeat domain 36
0.0391 PREDICTED: hypothetical protein LOC100130276
0.0123 PREDICTED: hypothetical protein LOC100132585
0.0446 small Cajal body-specific RNA 9 (SCARNA9), guide RNA
PREDICTED: hypothetical protein LOC132241, transcript variant
0.0004 1
0.0001 PREDICTED: hypothetical protein LOC100129211
0.0000 leiomodin 3 (fetal)
0.0004 FNPANH10 FNP cDNA, mRNA sequence
0.0410 microRNA 21 (MIR21), microRNA.
0.0005 hypothetical LOC791120, non-coding RNA.
0.0000 cDNA: FLJ22720 fis, clone HSI14320
0.0000 PREDICTED: similar to KIF27C
0.0287 golgi autoantigen, golgin subfamily a, 8A
0.0037 clock homolog (mouse)
0.0000 PREDICTED: hypothetical protein LOC648852
0.0001 hairy and enhancer of split 1, (Drosophila)
0.0374 Similar to Williams Beuren syndrome chromosome region 19
0.0250 small nucleolar RNA, C/D box 4A
0.0056 Similar to Golgi autoantigen, golgin subfamily A member 2
0.0012 somatostatin receptor 2
0.0003 aminomethyltransferase
0.0005 spectrin, beta, non-erythrocytic 5
0.0423 FNPARC07 FNP cDNA, mRNA sequence
0.0292 small nucleolar RNA, H/ACA box 32
0.0001 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
0.0000 cytochrome P450, family 26, subfamily B, polypeptide 1
0.0010 Similar to hypothetical protein MGC40405, transcript variant 1
0.0044 PREDICTED: hypothetical LOC645460, miscRNA
0.0180 Nedd4 binding protein 3
0.0019 PREDICTED: poly(A) binding protein, cytoplasmic 1-like
Supplemental Table 2S. miRNAs differentially expressed (>4-fold, p<0.05 following Ang II exposure (10-8M))
PCR Array Catalog:
MIHS-3001Z
Test Group:
Control Group:
Group 1
Control Group
Fold Difference:
p-value:
4
Arrays included in Test Group:
Arrays included in Control Group:
Genes Over-Expressed in
Gr 1 vs. Contr Group
Position
Mature ID
A03
hsa-let-7c
A04
hsa-let-7d-5p
A08
hsa-let-7g-5p
A12
hsa-miR-100-5p
A13
hsa-miR-101-3p
A15
hsa-miR-103a-3p
A21
hsa-miR-10a-5p
A23
hsa-miR-10b-5p
B10
hsa-miR-125a-5p
B11
hsa-miR-125b-5p
B17
hsa-miR-128
B23
hsa-miR-130a-3p
D01
hsa-miR-146a-5p
D04
hsa-miR-148a-3p
D13
hsa-miR-155-5p
D15
hsa-miR-15a-5p
D18
hsa-miR-16-5p
E18
hsa-miR-191-5p
F03
hsa-miR-194-5p
F04
hsa-miR-195-5p
F08
hsa-miR-196b-5p
F13
hsa-miR-19a-3p
F15
hsa-miR-19b-3p
F23
hsa-miR-204-5p
G07
hsa-miR-20b-5p
G09
hsa-miR-21-5p
G18
hsa-miR-218-5p
H09
hsa-miR-23b-3p
H11
hsa-miR-24-3p
H14
hsa-miR-26a-5p
H21
hsa-miR-28-5p
Sample 5, Sample 6, Sample 7
Sample 1, Sample 2, Sample 3
0.05
Fold Regulation
7.999
9.6311
14.5234
15.1508
8.8538
11.0614
38.4039
37.7826
5.641
4.3175
4.1804
9.1425
13.2799
12.7493
7.5026
10.894
55.3355
5.1739
5.0597
60.0453
5.8142
38.0872
25.639
4.9525
8.4185
6.7115
11.1799
12.108
6.6213
12.5757
10.8458
p-value
0.002638
0.01226
0.030977
0.001904
0.028837
0.006662
0.002925
0.001881
0.003671
0.001042
0.039235
0.040212
0.020504
0.008062
0.017022
0.006338
0.027317
0.025389
0.029365
0.026504
0.039988
0.035642
0.02162
0.006069
0.035778
0.012323
0.010885
0.020639
0.030616
0.015107
0.027642
I15
I21
J20
L01
N11
O09
O11
P10
P12
A01
A06
A07
A20
B13
C10
C15
D05
D09
D17
D16
E12
F10
H05
G22
H07
H15
I01
I03
I05
I07
I22
I23
J13
J17
K02
K04
K11
K16
K17
K22
L12
L23
M11
O15
P09
hsa-miR-30c-5p
hsa-miR-31-5p
hsa-miR-340-5p
hsa-miR-378a-5p
hsa-miR-542-3p
hsa-miR-652-3p
hsa-miR-7-5p
hsa-miR-99a-5p
hsa-miR-99b-5p
hsa-let-7a-5p
hsa-let-7e-5p
hsa-let-7f-5p
hsa-miR-107
hsa-miR-126-5p
hsa-miR-137
hsa-miR-140-5p
hsa-miR-148b-3p
hsa-miR-151a-5p
hsa-miR-15b-3p
hsa-miR-15b-5p
hsa-miR-18a-5p
hsa-miR-199b-3p
hsa-miR-224-5p
hsa-miR-22-5p
hsa-miR-23a-3p
hsa-miR-26b-5p
hsa-miR-29a-3p
hsa-miR-29b-3p
hsa-miR-29c-3p
hsa-miR-301a-3p
hsa-miR-31-3p
hsa-miR-32-5p
hsa-miR-337-5p
hsa-miR-33a-5p
hsa-miR-34a-5p
hsa-miR-34b-3p
hsa-miR-365b-3p
hsa-miR-374a-5p
hsa-miR-374c-5p
hsa-miR-377-3p
hsa-miR-424-5p
hsa-miR-454-3p
hsa-miR-495-3p
hsa-miR-744-5p
hsa-miR-98-5p
11.1775
5.1821
4.3998
4.2062
10.5226
6.152
7.9887
11.8331
8.0685
36.2119
34.4527
18.9942
4.2436
6.1198
5.331
8.8646
4.0557
9.5909
4.9592
27.0449
6.8671
8.3414
13.4055
5.3178
7.2468
34.974
4.1992
4.0142
4.4199
4.4841
14.776
7.1062
4.7345
7.3941
6.5368
9.1968
27.2569
15.5025
14.9342
4.0298
12.6225
9.6882
4.1468
6.498
20.9602
0.029498
0.006833
0.029632
0.00348
0.000923
0.005038
0.027465
0.012059
0.011272
0.056366
0.085361
0.070189
0.079621
0.086662
0.199723
0.120758
0.195599
0.154484
0.144357
0.203859
0.055695
0.072952
0.251133
0.096571
0.061058
0.09199
0.075161
0.6279
0.067073
0.270028
0.066365
0.058451
0.223248
0.672873
0.195792
0.374004
0.182755
0.125912
0.084957
0.616859
0.070351
0.12948
0.060901
0.190679
0.054446
Genes Under-Expressed in
Group 1 vs. Control Group
Position
B09
E20
F24
G02
G19
J02
K06
K12
K24
L16
L17
M21
N24
O14
P13
P14
A11
A16
A17
B02
B04
B05
B06
B14
B18
B19
B21
B22
C05
C06
C20
C21
C22
D11
D14
D24
E03
E05
E04
E06
E11
E15
E22
Mature ID
hsa-miR-125a-3p
hsa-miR-1914-3p
hsa-miR-205-5p
hsa-miR-206
hsa-miR-218-1-3p
hsa-miR-320b
hsa-miR-34c-3p
hsa-miR-370
hsa-miR-378a-3p
hsa-miR-4258
hsa-miR-429
hsa-miR-509-3p
hsa-miR-600
hsa-miR-720
cel-miR-39-3p
cel-miR-39-3p
hsa-miR-1
hsa-miR-105-5p
hsa-miR-106a-3p
hsa-miR-1207-5p
hsa-miR-122-3p
hsa-miR-1224-3p
hsa-miR-1224-5p
hsa-miR-1265
hsa-miR-1284
hsa-miR-129-1-3p
hsa-miR-129-2-3p
hsa-miR-129-5p
hsa-miR-133b
hsa-miR-134
hsa-miR-143-5p
hsa-miR-144-3p
hsa-miR-144-5p
hsa-miR-153
hsa-miR-155-3p
hsa-miR-181c-3p
hsa-miR-182-3p
hsa-miR-183-3p
hsa-miR-183-5p
hsa-miR-184
hsa-miR-187-5p
hsa-miR-18b-3p
hsa-miR-192-3p
Fold Regulation
p-value
-5.3287
-11.6172
-5.6565
-10.1174
-5.4826
-4.2411
-12.0269
-5.473
-5.3698
-6.073
-7.3218
-8.6417
-6.4729
-9.1451
-4.9459
-4.0663
-11.7659
-8.4077
-4.8047
-14.914
-9.6367
-21.6806
-12.9098
-6.2104
-6.133
-9.6367
-5.6627
-7.8897
-5.7026
-5.0034
-4.9597
-9.6367
-9.6367
-6.9866
-7.7225
-4.4664
-9.6367
-6.2508
-4.5576
-4.4959
-11.6157
-13.9526
-8.4831
0.026896
0.014646
0.040112
0.031606
0.043019
0.04052
0.021406
0.000839
0.02498
0.03664
0.017768
0.025546
0.004388
0.010625
0.132689
0.145846
0.07372
0.102381
0.131905
0.204135
0.09932
0.106026
0.156212
0.121453
0.066383
0.09932
0.129953
0.103627
0.123514
0.090402
0.147323
0.09932
0.09932
0.109837
0.11789
0.072645
0.09932
0.127495
0.122123
0.1283
0.121086
0.300198
0.101994
F17
F21
G01
G04
G12
G15
H02
H04
H08
H22
H23
I06
I08
I09
I10
I11
I12
J03
J06
J15
K05
K10
K14
K15
K18
L05
L15
L20
M01
M04
M05
M06
M07
M08
M13
M14
M15
M20
M23
M24
N01
N02
N03
N04
N05
N06
hsa-miR-200a-5p
hsa-miR-202-3p
hsa-miR-205-3p
hsa-miR-208b
hsa-miR-211-5p
hsa-miR-215
hsa-miR-222-5p
hsa-miR-223-5p
hsa-miR-23a-5p
hsa-miR-296-3p
hsa-miR-298
hsa-miR-300
hsa-miR-302a-3p
hsa-miR-302a-5p
hsa-miR-302b-3p
hsa-miR-302b-5p
hsa-miR-302c-3p
hsa-miR-323b-5p
hsa-miR-325
hsa-miR-338-5p
hsa-miR-34b-5p
hsa-miR-363-5p
hsa-miR-373-3p
hsa-miR-373-5p
hsa-miR-375
hsa-miR-383
hsa-miR-425-3p
hsa-miR-433
hsa-miR-483-5p
hsa-miR-486-3p
hsa-miR-486-5p
hsa-miR-488-3p
hsa-miR-489
hsa-miR-490-3p
hsa-miR-498
hsa-miR-499a-3p
hsa-miR-499a-5p
hsa-miR-508-5p
hsa-miR-512-5p
hsa-miR-513a-5p
hsa-miR-514a-3p
hsa-miR-517b-3p
hsa-miR-518b
hsa-miR-519d
hsa-miR-520f
hsa-miR-520g
-19.6682
-12.0895
-4.8158
-4.9288
-4.8384
-4.1096
-7.4862
-5.9167
-4.6836
-7.4087
-32.1319
-11.0419
-8.5752
-9.6367
-9.6367
-7.1813
-5.5931
-6.7385
-5.8797
-5.1311
-9.6367
-6.5945
-7.0096
-4.0312
-13.3325
-6.0715
-5.3741
-7.9413
-4.2324
-4.9581
-4.17
-9.6367
-12.5054
-9.8686
-8.151
-8.3219
-5.2903
-18.8221
-18.9986
-6.4791
-8.3842
-7.7762
-7.955
-9.6367
-5.6316
-11.3216
0.253733
0.104568
0.068411
0.05655
0.123745
0.141656
0.37209
0.116397
0.105251
0.115048
0.119368
0.248483
0.09788
0.09932
0.09932
0.102574
0.063362
0.362224
0.124776
0.111356
0.09932
0.113625
0.107579
0.053485
0.155636
0.16925
0.071938
0.099526
0.243308
0.135976
0.116803
0.09932
0.351276
0.192267
0.103123
0.06602
0.052293
0.237622
0.129276
0.134399
0.10238
0.104976
0.1039
0.09932
0.117168
0.126798
N07
N13
N15
N18
N20
N21
N22
O02
O03
O04
O05
O06
O07
O10
O13
O16
O18
O20
O22
O21
P08
hsa-miR-522-3p
hsa-miR-549a
hsa-miR-567
hsa-miR-575
hsa-miR-581
hsa-miR-583
hsa-miR-588
hsa-miR-606
hsa-miR-608
hsa-miR-622
hsa-miR-626
hsa-miR-639
hsa-miR-643
hsa-miR-661
hsa-miR-708-3p
hsa-miR-765
hsa-miR-802
hsa-miR-888-5p
hsa-miR-9-3p
hsa-miR-9-5p
hsa-miR-96-5p
-12.3435
-6.8545
-5.5068
-7.046
-6.7861
-9.6367
-4.8404
-9.6367
-5.4457
-10.3147
-4.9053
-6.4967
-6.8684
-6.9516
-6.1786
-10.2562
-7.2404
-6.2876
-9.6367
-5.1931
-9.6367
0.15577
0.174633
0.114559
0.123928
0.081973
0.09932
0.127276
0.09932
0.157874
0.103508
0.304322
0.17178
0.110013
0.086253
0.118698
0.070473
0.108158
0.095693
0.09932
0.122211
0.09932
Figure 1S. A radar graph depicting differentially expressed angiogenesis-related genes (p < 0.05) in combination with a
diagram showing expression shifts (microarray data) for their upstream regulators or downstream targets. Arrows –
stimulation. Shades of red – upregulation; Shades of blue – inhibition.
Figure 2S. Schematic diagram of interactions between STAT3 and miRNAs found to be diferentially expressed in
present study.