Supplementary Online Content
Serum microRNA expression profile as a biomarker in the diagnosis and
prognosis of pancreatic cancer
Supplementary Methods
RNA ISOLATION AND RT-qPCR ASSAY
Total RNA was extracted from 100 μl of serum by phenol/chloroform purification and
centrifugation in isopropyl alcohol. Briefly, 100 μl of serum was mixed with 300 μl of
RNase-free water, 200 μl of phenol, and 200 μl of chloroform. The sample was
vortexed and incubated at room temperature for 15 min. The mixture was centrifuged
at 12,000 g for 10 min and the upper aqueous layer was collected. Subsequently, a
1/10 volume of 3 M sodium acetate and a two-fold volume of isopropyl alcohol were
added to the aqueous phase. The total RNA was precipitated by incubation at -20 C
for 1 h. The RNA pellet was collected by centrifugation at 16,000 g for 20 min,
washed once with 75% ethanol, dried for 10 min at room temperature, and then
dissolved in 20 μl of RNase-free water.
TaqMan probe-based RT-qPCR assay was carried out using a commercial kit
(Applied Biosystems, Foster City, CA, USA). Briefly, 2 μl of total RNA was reverse
transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and
the stem-loop RT primer (Applied Biosystems, Foster City, CA, USA). Real-time
PCR was performed using TaqMan miRNA probes on an Applied Biosystems 7300
Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The
threshold cycle (Cq) values were determined using the fixed threshold settings. All
reactions, including no-template controls, were performed in triplicate. To calculate
the absolute expression levels of target miRNAs, a series of synthetic miRNA
oligonucleotides of known concentrations were also reverse transcribed and amplified
to generate a standard curve. It was linear on a semi-logarithmic plot in a range from
10 fM to 105 fM. The absolute amount of each miRNA was then calculated by
referring to the standard curve. Because U6 and 5S rRNAs were degraded in the
serum samples, and there was no current consensus on housekeeping miRNAs for the
RT-qPCR analysis of serum miRNAs, the expression levels of miRNAs were directly
normalized to the serum volume in this study.
SEQUENCING BY SYNTHESIS TECHNOLOGY
Serum samples from 25 PaC patients and 25 healthy subjects were pooled separately,
and total RNA was extracted from the serum pool using the TRIzol Reagent
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After
poly-acrylamide gel electrophoresis (PAGE) purification of RNA molecules smaller
than 30 base pairs (bp), a pair of adaptors was ligated to the 5’ and 3’ ends of the
RNAs. The small RNA molecules were amplified using the adaptor primers for 17
cycles, and fragments of around 90 bp (small RNA + adaptors) were isolated from
PAGE gels. The purified DNA was directly supplied for the cluster generation and
sequencing analysis using an Illumina Genome Analyzer (Illumina, San Diego, USA)
according to the manufacturer’s instructions. Image files generated by the sequencer
were processed to produce digital-quality data. The subsequent procedures included
summarizing data production, evaluating sequence quality and depth, calculating
1
length distribution of small RNAs and filtering contaminated reads. After masking the
adaptor sequences, the clean reads were aligned against the miRBase database
16.0 by the Smith-Waterman algorithm. Finally, the total sequencing frequency of
each sample was adjusted to an equal scale of 1,000,000.
For comparison of the expression patterns of serum miRNAs between PaC cases
and controls, a miRNA was considered “significantly altered” only if at least 20
copies were detected by sequencing in either the control or the PaC group, and an at
least two-fold alteration in expression was observed in the PaC group compared to
that in the control group.
SERUM CA19-9 AND CEA DETERMINATION
The serum values of CA19-9 and CEA were measured using commercial kits
(CA19-9 RIA, Abbott AxSYM System, Chicago, IL; CEA fluoro-immunometric assay,
Beckman Coulter Inc, Fullerton, CA). The upper limits of normal values for CA19-9
and CEA were 37 U/ml and 5 U/ml, respectively.
SURVIVAL ANALYSIS
Clinical follow-up examinations were based on periodic visits for the stage III and IV
patients (every three months during the first year and six months during the second
year). Expression levels of these seven miRNAs in PaC were stratified by the median
value, and the association between survival outcome and miRNA levels was
determined using the Kaplan-Meier method. We also calculated the level of statistical
significance for each miRNA, based on a univariate and multivariate Cox proportional
hazard regression model.
2
Supplementary Tables
Supplementary Table 1: The final pathologic diagnosis results of the 55 suspicious
PaC cases.
Supplementary Table 2: Differentially-expressed miRNAs in PaC serum samples
compared to those in control serum samples determined by sequencing technology.
Supplementary Table 3: Differentially-expressed miRNAs in PaC serum samples
compared to those in control serum samples in the training set.
Supplementary Table 4: Survival analysis of PaC patients in relation to
clinicopathological characteristics and miRNA expression.
Supplementary Table 5: The forecast accuracy rate of the serum 7-miRNA profile,
CA19-9, and CEA as a PaC biomarker for samples in double-blind test.
Supplementary Table 6: The forecast accuracy rate of the serum 7-miRNA profile,
CA19-9, and CEA as a PaC biomarker for cases suspected of having PaC.
Supplementary Table 7: Differentially-expressed miRNAs in stage I PaC serum
samples compared to those in control serum samples.
Supplementary Table 8: The limit of detection and dynamic range for each miRNA
3
Supplementary Table 1
Pathologic diagnosis
Age (years)
Male
Female
PaC (n=46)
Pancreatic pseudocyst (n=4)
Duodenal adenocarcinoma (n=3)
Cholangiocarcinoma (n=1)
59.11±10.25
47.75±16.60
60.33±13.65
34
63
28
3
2
1
1
18
1
1
Hepatocellular carcinoma ( n=1）
4
Supplementary Table 2
Up-regulated miRNAs in PaC serum samples
compared to control serum samples
order
name
Reads
Reads
in
in PaC
control
1
hsa-let-7a
649
1566
2
hsa-let-7b
381
2454
3
hsa-let-7c
202
3808
4
hsa-let-7d
119
875
5
hsa-let-7f
126
2092
6
hsa-let-7g
33
458
7
hsa-let-7i
19
962
8
hsa-miR-100
11
624
9
hsa-miR-101
5
844
10
hsa-miR-103
55
476
11
hsa-miR-122
1438
31232
12
hsa-miR-125a-5p
19
134
13
hsa-miR-128
16
59
14
hsa-miR-140-3p
48
266
15
hsa-miR-148a
1
193
16
hsa-miR-185
362
873
17
hsa-miR-185*
0
135
18
hsa-miR-191
29
846
19
hsa-miR-192
14
14894
20
hsa-miR-193b*
0
185
21
hsa-miR-199a-3p
16
1604
22
hsa-miR-20a
0
245
23
hsa-miR-21
38
1571
24
hsa-miR-210
2
81
25
hsa-miR-215
0
368
26
hsa-miR-24
21
172
27
hsa-miR-25
21
237
28
hsa-miR-26a
14
227
29
hsa-miR-27a
7
240
30
hsa-miR-27b
4
462
31
hsa-miR-29a
47
1800
32
hsa-miR-29c
7
232
33
hsa-miR-30a
4
1679
34
hsa-miR-30d
187
926
35
hsa-miR-320a
361
1193
36
hsa-miR-320b
9
188
5
Down-regulated miRNAs in PaC serum
samples compared to control serum samples
order
name
Reads
Reads
in
in PaC
control
1
hsa-miR-1
229
15
2
hsa-miR-107
35
3
3
hsa-miR-125b
21
0
4
hsa-miR-139-3p
68
0
5
hsa-miR-146b-5p
26
5
6
hsa-miR-150
151
0
7
hsa-miR-197
49
0
8
hsa-miR-206
203
0
9
hsa-miR-22
481
0
10
hsa-miR-221
41
0
11
hsa-miR-222
30
2
12
hsa-miR-28-3p
38
0
13
hsa-miR-339-5p
78
0
14
hsa-miR-423-5p
585
152
15
hsa-miR-484
53
0
16
hsa-miR-486-3p
24
0
17
hsa-miR-486-5p
1640
9
18
hsa-miR-532-3p
26
0
19
hsa-miR-584
22
0
37
38
39
40
41
42
43
44
hsa-miR-361-5p
hsa-miR-378
hsa-miR-451
hsa-miR-483-5p
hsa-miR-92a
hsa-miR-95
hsa-miR-99a
hsa-miR-532-5p
1
26
14
70
115
0
40
0
322
382
34
1130
331
99
413
377
6
Supplementary Table 3
order
name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
let-7a
let-7b
let-7c
let-7d
let-7f
let-7i
miR-1
miR-20a
miR-21
miR-22
miR-24
miR-25
miR-26a
miR-27a
miR-27b
miR-28-3p
miR-29a
miR-29c
miR-30a
miR-30d
miR-92a
miR-95
miR-99a
miR-100
miR-101
miR-103
miR-107
miR-122
miR-125a-5p
miR-125b
miR-128
miR-139-3p
miR-140-3p
miR-146-5p
miR-148a
miR-150
miR-185
miR-185*
miR-191
miR-192
mean fold
change
p-value
1.0943746
0.978667
1.789553
0.586468
1.6789
1.789564
3.13
4.24
3.64E-07
2.28E-05
2.67
0.001255
5.08
8.18E-10
4.16
0.128493
1.2960753 0.008085
1.07
0.7567261
1.1431785
1.320792
1.4954582
0.8956235
1.2414502
1.1179528
2.69
1.0234521
1.079218
1.0661197
1.094375
3.26
0.3623874
0.1492103
0.0731187
0.5688125
0.3052584
0.5932393
2.61E-05
0.9108538
0.7229841
0.7972754
0.586468
0.104824
1.3597159 0.2975648
1.0538031 0.8654724
1.053803 0.865472
1.042287 0.7037122
1.0481299 0.8971156
1.746494 0.057758
2.1
0.007051
2.87
0.000372
1.3246894 0.2644458
7
non-significant
non-significant
non-significant
undetectable
undetectable
undetectable
undetectable
significant
significant
undetectable
significant
significant
non-significant
non-significant
non-significant
Cq value > 35
non-significant
non-significant
non-significant
non-significant
non-significant
non-significant
significant
non-significant
non-significant
non-significant
non-significant
non-significant
undetectable
non-significant
non-significant
non-significant
non-significant
undetectable
non-significant
non-significant
significant
undetectable
significant
non-significant
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
miR-193b*
miR-197
miR-199a-3p
miR-206
miR-210
miR-215
miR-221
miR-222
miR-320a
miR-320b
miR-339-5p
miR-361
miR-378
miR-411
miR-423-5p
miR-484
miR-451
miR-483
miR-486-3p
miR-486-5p
miR-532-5p
miR-532-3p
miR-584
1.117953
1.363585
0.593239
0.2153148
1.3872444 0.3868377
0.9732919 0.9444299
0.9764046 0.8690469
1.493328 0.0640379
1.5194114 0.3374292
1.2340253 0.4139875
1.796262 0.007805
1.7464941 0.0577575
5.632765 0.1209546
1.7962625 0.0078049
8
Cq value > 35
non-significant
non-significant
undetectable
non-significant
non-significant
undetectable
non-significant
non-significant
Cq value > 35
undetectable
non-significant
non-significant
non-significant
undetectable
Cq value > 35
non-significant
non-significant
undetectable
undetectable
non-significant
undetectable
Cq value > 35
Supplementary Table 4
Variable
Subset
Univariate analysis
Gender
male / female
Age
age < 48 / age ≥ 48
TNM stage
IV / III
high / low
miR-20a
high / low
miR-21
high / low
miR-24
high / low
miR-25
high / low
miR-99a
high / low
miR-185
high / low
miR-191
Multivariate analysis
Gender
male / female
Age
age < 48 / age ≥ 48
TNM stage
IV / III
high / low
miR-20a
high / low
miR-21
high / low
miR-24
high / low
miR-25
high / low
miR-99a
high / low
miR-185
high / low
miR-191
9
Hazard ratio (95% CI)
p-value
1.50 (0.67-3.34)
0.54 (0.24-1.21)
0.92 (0.38-2.24)
0.56 (0.24-1.34)
2.89 (1.22-6.81)
0.73 (0.32-1.65)
0.69 (0.31-1.57)
1.48 (0.67-3.30)
1.09 (0.48-2.46)
1.50 (0.67-3.34)
0.32
0.14
0.86
0.20
0.02
0.45
0.39
0.33
0.83
0.32
1.50 (0.67-3.34)
0.46 (0.18-1.18)
1.12 (0.35-3.49)
0.53 (0.17-1.64)
8.77 (2.00-38.39)
0.52 (0.17-1.52)
0.79 (0.30-2.02)
0.64 (0.15-2.65)
0.46 (0.13-1.62)
1.15 (0.29-4.49)
0.32
0.11
0.84
0.28
0.00
0.23
0.62
0.54
0.23
0.84
Supplementary Table 5
Pathologic diagnosis
serum 7-miRNA
biomarker
CA19-9
CEA
PaC (n=77)*
control (n=52)**
Accuracy rate***
77
52
100%
68
44
86.8%
47
22
51
50
76.0%
55.8%
*
PaC-positive
**
PaC-negative
***
Accuracy rate = (PaC-positive + PaC-negative) / total
Supplementary Table 6
Other diseases
(n=9)**
9
6
4
4
PaC (n=46)*
Pathologic diagnosis
serum 7-miRNA biomarker
CA19-9
CEA
46
40
27
16
*
PaC-positive
**
PaC-negative
***
Accuracy rate = (PaC-positive + PaC-negative) / total
10
Accuracy rate***
100%
83.6%
56.4%
36.4%
Supplementary Table 7
miR-20a
miR-21
miR-24
miR-25
miR-99a
miR-185
miR-191
Normal Control
(n=158)
61.46±4.63
10.68±1.31
28.24±2.91
6.12±0.84
9.87±1.15
11.50±0.91
39.13±4.07
stage I PaC
(n=26)
221.02±36.48
43.96±11.21
71.10±14.18
36.43±6.37
47.49±6.60
81.83±22.83
139.38±22.52
mean fold
change
3.70
4.58
2.63
6.57
5.79
6.55
4.03
p-value
1.08×10-15
1.47×10-09
4.28×10-06
6.25×10-18
3.62×10-19
2.35×10-12
5.64×10-12
Supplementary Table 8
The limit of detection and dynamic range for each miRNA (fmol/L)
limit of detection (fmol/L)
dynamic range (fmol/L)
miR-20a
10
10-105
miR-21
10
10-106
miR-24
10
10-105
miR-25
10
10-105
miR-99a
10
10-106
miR-185
10
10-106
miR-191
10
10-106
11
Supplementary Figures
Supplementary Figure 1. Evaluation of the RNA extraction efficiency and
reproducibility of RT-qPCR assay. A) The representative standard curve for synthetic
single-strand miRNA (synthesized by TaKaRa, Dalian, China). For each assay, a
standard calibration curve was prepared by a ten-fold serial dilution of miRNA from
10 fM to 107 fM, and the level of miRNA was assessed by the RT-qPCR assay. The
resulting Cq values were plotted versus the log10 of the amount of input miRNA. Each
point represents the mean of four independent experiments. B) Comparison of the
expression levels of 13 miRNAs in fresh serum samples and serum samples existed at
room temperature over night. Serum collected from a single subject was divided into
two samples of equal volume. One was analyzed immediately while the other was
analyzed until existed at room temperature over night. The Cq values from two assays
were plotted against each other, and then the data was fit to a straight line. C)
Comparison of the expression levels of 15 miRNAs in duplicate samples. The
expression levels of 15 miRNAs in two RNA samples isolated from equal volume of
serum of a same subject were characterized using the RT-qPCR assay. The Cq values
from the replicate assays were plotted against each other, and then the data was fit to a
straight line.
Supplementary Figure 2. The expression level of the serum 7-miRNA signature in
PaC cases. A-G) Differential expression of the seven miRNAs in the serum of PaC
cases (n = 120) compared to that in controls (n = 106) or CP cases (n = 82). The
expression levels of the seven serum miRNAs were measured by TaqMan
probe-based RT-qPCR. Note that the expression levels of the seven serum miRNAs
were significantly higher in the PaC patients than those in the normal controls and CP
patients. H-J) Differential expression of the seven miRNAs in PaC cases with
different stage. PaC cases were stratified by tumor stage (III and IV vs I and II). Mean
risk score (H) and the expression levels of miR-21 and miR-24 (I and J) in each group
were shown. (*, p < 0.05; **, p < 0.01).
Supplementary Figure 3. Cluster analysis of the miRNA differentially expressed
between PaC and control serum samples. A and B) For training set (25 cases and 25
controls) and validation set (95 cases and 81 controls), the absolute expression values
of serum miRNAs in PaC cases and controls were normalized, mean-centered,
clustered and plotted as a heat map. C) Cluster analysis of the miRNA differentially
expressed between PaC and CP cases (120 PaC cases and 82 CP cases). D) Cluster
analysis of the miRNA differentially expressed between CP cases and controls (82 CP
cases and 106 controls).
Supplementary Figure 4. Comparison of the expression levels of 7 miRNAs in PaC
and normal adjacent tissue (NAT) samples. The relative expression of 7 miRNAs
(PaC vs. NAT), normalized to U6, was assessed using the RT-qPCR assay (n = 3). For
comparison, the expression levels of miRNAs in NAT samples were arbitrarily set at 1.
The y-axis shows arbitrary units representing relative miRNA levels. The results are
presented as the mean ± SE. **, p < 0.01; *, p < 0.05.
12
Supplementary Figure 1
13
Supplementary Figure 2
14
Supplementary Figure 3
15
Supplementary Figure 4
16