Supplementary Material
MicroRNA Deregulation and Pathway Alterations in Nasopharyngeal Carcinoma
1
Hua-Chien Chen, 1Gian-Hung Chen, 1Yi-Hsuan Chen, 1Wen-Ling Liao, 1ChienYuan Liu, 2Kai-Ping Chang, 1Yu-Sun Chang and 1Shu-Jen Chen*
Content
Material and Methods
Figure legend
Figure S1
Table S1
page 1
page 3
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page 5
Material and Methods
Western blot analysis
Protein extracts, separated by SDS-PAGE and transferred onto PVDF membranes, were
probed with antibodies against CCNE2 (SC-28351, 1:5000, Santa Cruz Biotechnology,
Santa Cruz, CA) or actin (MAB1501, 1:5000, Chemicon, Billerica, MA). Proteins of
interest were detected with HRP-conjugated sheep anti-mouse IgG antibody (1: 5000, GE
Healthcare, Uppsala, Sweden) and visualized with the Pierce ECL Western blotting
substrate (Thermo Scientific, Rockford, IL), according to the provided protocol.
Immunohistochemistry
Tissue sections were fixed with 10% formaldehyde, embedded in paraffin and cut into 4
mm-thick sections. Staining for CCNE2 was carried out using the Envision-kit (DAKO,
Carpinteria, CA). Briefly, the sections were deparaffinized with xylene, dehydrated with
ethanol and then heated in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase
activities were inactivated in 3% H2O2 for 10 min at room temperature, and the sections
were blocked with 3% normal goat serum in 0.2 M PBS (pH 7.4). Samples were then
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incubated with anti-CCNE2 at room temperature for 1 hour. Secondary anti-mouse
antibody-coated polymer peroxidase complexes (DAKO) were then applied for 30 min at
room temperature, followed by treatment with substrate/chromogen (DAKO) and a
further incubation for 5–10 min at room temperature. Slides were counterstained with
hematoxylin.
Construction and Transfection of Lentiviral vectors with miR-9
Double-stranded oligonucleotide encoding miR-9 precursor or negative control
(scrambled sequence) were synthesized and inserted into pcDNA6.2-GW/Em-GFP-miR
expression vector (InVitrogen, Carlsbad, CA) which contains human CMV promoter.
The miR-9 and negative control expression cassette was transferred into the lentiviral
expression plasmid (pLenti6/V5-DEST) with the Gateway recombination technology
using the pDONR221 vector as an intermediate vector. To generate lentiviral particles,
the miR-9 or negative control lentiviral expression vector were co-transfected with
packaging vectors (ViraPower packaging Mix, InVitrogen) into HEK293-FT cells using
lipofectamine 2000 (InVitrogen). Culture supernatants were harvested on day 3 and used
to infect HK1 cells.
Cell culture
Human NPC cell line, HK1, was cultured in RPMI-1640 (InVitrogen) with 10% fetal calf
serum at 37 degree in a humidified chamber containing 5% CO2. HEK293-FT cells were
maintained DMEM (InVitrogen) supplemented with 10% fetal calf serum and 0.5 mg/L
G-418.
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Figure Legend
Figure S1. MicroRNA expression patterns distinguish normal from NPC tissues. (A).
Unsupervised hierarchical clustering of 223 miRNAs in 7 normal (blue)-NPC (red)
paired tissues. The hierarchical clustering was performed using squared Euclidean as
distance measure and Ward’s method for linkage analysis. miRNA levels were expressed
as 39 – Ct after normalized to the geometric mean of miR-103 and miR-191 (Peltier &
Latham, 2008). (B). Selection of miRNAs differentially expressed in 7 paired normalNPC tissues. Differentially expressed miRNAs were selected based on t-test (p < 0.01)
and median fold change (≥ 3 fold). Seven miRNAs showed significant up-regulation and
27 miRNAs showed significant down-modulation in NPC samples. (C). Principle
component analysis using the expression levels of 34 miRNAs in 9 normal (blue) and 13
NPC (red) samples. (D). Unsupervised hierarchical clustering of 34 differentially
expressed miRNAs in normal (blue) and NPC (red) samples. The hierarchical clustering
was performed using Pearson’s dissimilarity as distance measure and Ward’s method for
linkage analysis. MiRNA levels were expressed after standardization.
Peltier HJ, Latham GJ (2008) Normalization of microRNA expression levels in
quantitative RT-PCR assays: identification of suitable reference RNA targets in normal
and cancerous human solid tissues. RNA 14: 844-52
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Figure S1
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Table s1. microRNAs significantly altered in NPC tissues
microRNA
Chromosome
Mean Expression*
Normal
NPC
Fold change
T-test
p-value
Mann-Whitney
Up-regulated
miR-196b
7p15
4.00
6.52
5.72
5.23E-05
miR-138
3p21, 16q13
4.54
7.04
5.67
1.61E-04
miR-155
21q21
7.58
9.89
4.93
1.23E-04
miR-18a
13q31
5.14
7.27
4.41
2.74E-04
miR-142-3p 17q22
9.54
11.54
4.01
2.72E-04
miR-25*
7q22
7.30
9.07
3.39
2.11E-04
miR-205 a
1q32
16.17
17.88
3.26
7.40E-04
miR-106a
Xq26
9.36
10.99
3.1
7.43E-04
miR-17* b
13q31
5.08
6.60
2.87
2.38E-03
miR-15b* b 3q26
4.65
6.16
2.85
2.10E-04
miR-17 b
13q31
10.78
12.22
2.72
2.38E-03
down-regulated
miR-204
9q21
10.48
4.79
-51.62
1.22E-07
miR-449a
5q11
8.64
4.27
-20.67
4.64E-06
miR-34c-3p 11q23
12.05
7.93
-17.32
6.74E-04
miR-187
18q12
10.34
6.57
-13.62
1.26E-06
miR-34c-5p 11q23
9.41
5.65
-13.55
9.19E-04
miR-145
5q33
16.94
13.22
-13.2
6.37E-05
miR-143
5q33
12.63
8.95
-12.77
6.63E-05
miR-34b
11q23
8.45
5.19
-9.57
1.81E-03
miR-100
11q24
13.06
9.84
-9.32
2.03E-03
miR-139-5p 11q13
11.71
8.62
-8.53
1.80E-05
miR-148a
7p15
8.22
5.22
-8.02
1.72E-04
miR-195
17p13
10.09
7.11
-7.91
2.85E-04
miR-30a*
6q13
10.19
7.27
-7.57
1.76E-03
miR-9
1q22, 5q14, 15q26
7.24
4.35
-7.44
9.22E-05
miR-497
17p13
12.25
9.42
-7.09
5.07E-04
miR-130a
11q12
10.04
7.61
-5.39
8.45E-04
miR-135a
3p21, 12q23
7.97
5.55
-5.35
5.16E-03
miR-31
9p21
12.66
10.31
-5.1
1.78E-04
miR-199b-5p 9q34
7.76
5.49
-4.84
4.62E-03
miR-29c
1q32
7.87
5.85
-4.08
5.25E-03
miR-152
17q21
10.41
8.44
-3.93
3.79E-03
miR-200a
1p36
7.40
5.43
-3.93
1.00E-03
miR-200b
1p36
12.13
10.16
-3.92
4.62E-03
miR-532-3p Xp11
13.66
11.73
-3.81
2.77E-04
miR-660 c
Xp11
9.64
7.77
-3.64
1.47E-03
miR-26a c
3p22
13.51
11.70
-3.51
7.56E-06
miR-449b c 5q11
5.56
3.96
-3.03
5.55E-03
* Expressed as 39 - Ct after normalized to geometric mean of miR-103 and miR-191
a
Mann-Whitney test P-value > 0.01, not inculded in hierarchical clustering in figure s1d
b
Fold change < 3, not included in hierarchical clustering in figure s1d
c
not included in gene set listed in Table 1
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1.39E-04
6.73E-04
1.32E-04
1.32E-03
3.69E-03
1.13E-03
1.16E-02
1.19E-03
3.54E-03
9.48E-04
1.46E-02
1.22E-07
4.64E-06
6.74E-04
1.26E-06
9.19E-04
6.37E-05
6.63E-05
1.81E-03
2.03E-03
1.80E-05
1.72E-04
2.85E-04
1.76E-03
9.22E-05
5.07E-04
8.45E-04
5.16E-03
1.78E-04
4.62E-03
5.25E-03
3.79E-03
1.00E-03
4.62E-03
2.77E-04
4.50E-03
1.20E-04
4.50E-03