IJBM

Int J Biol Markers 2015; 30 (2): e226-e233

DOI: 10.5301/jbm.5000134

eISSN 1724-6008 ORIGINAL ARTICLE

Identification of biomarkers for the prognosis of

pancreatic ductal adenocarcinoma with miRNA
microarray data
Song Wang, Yanxun Zhao, Dongsheng Li, Liangchen Zhu, Zugang Shen

Department of Emergency Surgery, Tongji Hospital, Shanghai - PR China

ABSTRACT
Background: The aim of this study was to explore the mechanism of chemotherapy resistance and to screen bio-
markers of pancreatic ductal adenocarcinoma (PDAC).
Methods: MicroRNA (miRNA) expression profile data for GSE38781 were downloaded from the Gene Expression
Omnibus database. Differentially expressed miRNAs between short–overall survival (OS) and long-OS patients
were screened with the limma package in R. The function and protein–protein interaction (PPI) network of the
miRNA target genes were further investigated. Finally, multivariate statistical analysis was performed to verify the
significant miRNAs obtained in our work.
Results: In total, 66 miRNAs were identified to be differentially expressed. Gene ontology (GO) and pathway
enrichment analysis showed that 163 miRNA target genes were mainly enriched in heart function, cancer devel-
opment and angiogenesis. Ten nodes, including TGFBR1, TGFBR2, ACVR1 and SHC1, were found to be hub nodes
in the PPI network. Multivariate statistical analysis showed 8 of the most significant miRNAs could completely
distinguish the 2 groups of samples. Seven target genes (i.e., RET, ETS1, RHOA, NUMB, TIAM, ITGA5 and YY1) of
the 8 significant miRNAs were found to be associated with control of cell fate decisions, T-cell lymphoma invasion
and angiogenesis enhancement.
Conclusions: The heart function–related pathway, cell cycle, immune system and angiogenesis may be dysregu-
lated in patients with poorer prognosis. The significant nodes (e.g., TGFBR1, TGFBR2, ACVR1 and SHC1) in the PPI
network may be potential biomarkers for predicting outcomes for patients with pancreatic cancer. The significant
miRNAs and gene targets may be potential biomarkers or therapeutic targets for PDAC.
Keywords: Differentially expressed microRNAs, Hierarchical clustering analysis, Multivariate statistical analysis,
Pancreatic adenocarcinoma, Protein-protein interaction network

Introduction to have a poor prognosis, which partly results from the ab-
sence of symptoms in its early stages. It is reported that the
Pancreatic cancer is the fourth leading cause of cancer- median survival time of pancreatic adenocarcinoma patients
related death in Western countries (1, 2). Pancreatic adeno- is less than 6 months, and 5% of patients have a dismal, 5-year
carcinomas characterized by glandular architecture accounted survival (4, 5).
for 95% of all pancreatic tumors in 2011 (3). Pancreatic adeno- At present, the general treatments for pancreatic adeno-
carcinoma can result in various symptoms, such as abdominal carcinoma primarily include surgery, radiation and chemother-
pain, significant weight loss, poor appetite and jaundice. Pa- apy. Radiation and chemotherapy are used for patients not
tients with pancreatic adenocarcinoma are commonly found suitable for resection with curative intent, or as adjuvant treat-
ment to increase the survival of patients with surgical resection
(6). However, the prognosis of patients after complete resec-
Received: August 1, 2014 tion is poor, with a 3-year disease-free survival rate of 27% and
Accepted: December 17, 2014 median overall survival (OS) of 15-19 months (7). Therefore, an
Published online: March 16, 2015 increasing number of studies have attempted to make contri-
butions to discovering novel biomarkers with good sensitivity
Corresponding author: for adjuvant therapy and prognosis prediction.
Zugang Shen MicroRNAs (miRNAs) are a group of small noncoding RNA
Department of Emergency Surgery
Tongji Hospital molecules, which possess functions related to transcription
Road of Xin Cun 389 and posttranscriptional regulation of gene expression (8).
Shanghai 200065, PR China The miRNAs are expressed differentially in normal and malig-
shenzugang@163.com nant tissues, and tumor-associated miRNAs are detectable in

© 2015 Wichtig Publishing

Wang et al
patient circulation (9). Investigations of miRNAs have pro-
vided additional insights for the exploration of new therapies
and prognosis information for cancers. It is reported that
microRNA-26b targeting ubiquitin-specific peptidase 9 (US-
P9X) can prevent hepatocellular carcinoma by inhibiting the
epithelial-mesenchymal transition (10). MiRNA-15b has been
found to be a potential biomarker for predicting the antitu-
mor effect of rosemary in colorectal and pancreatic cancers
(11). However, information regarding miRNAs in pancreatic
adenocarcinoma is insufficient.
In the present study, we downloaded the miRNA expression
profiling of patients with pancreatic ductal adenocarcinoma
(PDAC) from the Gene Expression Omnibus (GEO) database.
The differentially expressed miRNAs between patients with
short OS and long OS were investigated. The purpose of this pa-
per was to explore the relationship of tumor-associated miRNAs
with chemotherapy sensitivity and prognosis.
Materials and Methods
Affymetrix microarray data and differentially
expressed miRNA analysis
The miRNA expression profiling of GSE38781 was down-
loaded from the publicly available GEO database (12), which
had been deposited by Giovannetti and his colleagues (7).
Patients with PDAC who underwent radical surgical resec-
tion with curative intent (pancreaticoduodenectomy, to-
tal pancreatectomy and distal pancreatectomy) and who
were treated with 3 cycles of standard gemcitabine adju-
vant regimen were carefully selected. A total of 26 tumor
tissues were collected from patients with PDAC, among
which, 19 samples were unsuitable for miRNA analysis
and were used for the microarray development. The 19
samples were divided into 2 groups including a short-OS
group (from PDAC patients dying within 1 year of diagnosis)
and long-OS group (from patients who had survived more
than 30 months). The raw data were downloaded for fur-
ther studies based on the platform of GPL14903 (3D-Gene
Human miRNA V16_1.0.0).
After the raw data were preprocessed, the differentially
expressed miRNAs between the short-OS and long-OS group
were analyzed with the limma package in R (13). A multi-
ple testing correction was performed using the Benjamini-
Hochberg false discovery rate (HB FDR) (14). An FDR <0.05
and |log FC (fold change)| >1 were used as the threshold for
identifying differentially expressed miRNAs.
Hierarchical clustering analysis
Hierarchical clustering has been widely applied in ana-
lyzing gene expression patterns (15, 16). With hierarchical
clustering, the most similar expression patterns can be clus-
tered in a hierarchy of nested subsets. The expression values
of differentially expressed miRNAs were selected according
to the probe information from the downloaded files. The
hierarchical clustering analysis for differentially expressed
miRNAs was performed with Cluster software (17), and the
results were visualized on a heatmap with Treeview soft-
ware (18).
© 2015 Wichtig Publishing
e227
MiRNA target gene prediction and function
enrichment analysis
To analyze the role of miRNAs in biological mechanism in
response to chemotherapy, a gene ontology (GO) function
analysis was performed for target genes of the differentially
expressed miRNAs. The target genes for differentially ex-
pressed miRNAs were collected based on the databases of
miRecord (19), miRTarBase (20) and Tarbase 6.0 (21). GO and
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
enrichment analysis for the miRNA target genes were per-
formed using the Database for Annotation, Visualization and
Integrated Discovery (DAVID) tool (22). A p value <0.05 was
defined as the cutoff value.
Construction of protein–protein interaction network
To further analyze the function of miRNA target genes
in PDAC patients, we performed a protein–protein interac-
tion (PPI) network analysis. The miRNA target genes which
were enriched in at least 1 pathway or GO term were input
into the Human Protein Reference Database (HPRD; http://
www.hprd.org/) (23), and the protein pairs involved with
the target genes were obtained. The miRNA target genes
and those normal genes that were interacting with at least
3 target genes were mapped into the PPI network using the
Python program (24). The PPI network was visualized with
the Cytoscape package (25).
Furthermore, to identify the significant nodes, topo-
logical characteristics of the PPI network were examined
by a Cytoscape plug-in, including for node degree, average
shortest path length (ASPL), clustering coefficient (CC) and
centrality. The significant node genes were further analyzed
based on the automatically selected weighting method with
the GeneMANIA tool (26).
Multivariate statistical analysis
To verify the significance of the miRNAs identified in this
paper, we performed logistic regression analysis for the dif-
ferentially expressed miRNAs, using 2 multivariate statistical
methods: logistic regression analysis and discriminant analysis.
Logistic regression analysis was performed on the miRNA
expression values in 19 patients to identify the miRNAs that
could effectively distinguish the samples from the 2 groups
(short-OS vs. long-OS groups). Discriminant analysis, also
known as “resolution,” is a multivariate statistical analysis to
identify the type ascription according to various characteris-
tic values of a study in certain conditions. Two kinds of mul-
tivariate statistical analysis were performed using SPSS 19.0
software with a threshold of p<0.05.
Results
Identification of differentially expressed miRNAs and hierar-
chical clustering analysis
To find the differentially expressed miRNAs between the
short-OS group and long-OS group, we obtained the publicly
available microarray dataset GSE38781 from the GEO database.
e228 Chemotherapy resistance analysis

Function enrichment analysis

After searching in the databases of miRecord, miRTarBase

and Tarbase 6.0, we obtained 163 target genes for 11 differ-
entially expressed miRNAs. GO enrichment and KEGG path-
way analysis were performed for these genes with DAVID.
As shown in Table I, the significantly enriched pathways
were mainly related to cancer processes and cardiac func-
tion, such as colorectal cancer, pancreatic cancer, arrhyth-
mogenic right ventricular and hypertrophic cardiomyopathy.
The most significant pathway was the hsa05210:colorectal
cancer pathway (p = 2.47E-04). The overexpressed GO terms
were relevant for regulation of transcription, regulation
of RNA metabolic process, cell motion, cell migration and
blood vessel development (Tab. I).

TABLE I - F unction enrichment analysis of target genes for differen-

tially expressed miRNAs

Term Count % p Value

GO enrichment analysis
 GO:0006355: regulation of 43 26.875 7.56E-07
transcription, DNA-dependent
 GO:0006357: regulation of 25 15.625 1.27E-06
transcription from RNA
 GO:0051252: regulation of RNA 43 26.875 1.37E-06
metabolic process
 GO:0045449: regulation of 51 31.875 2.27E-05
transcription
 GO:0006350: transcription 40 25 5.32E-04
 GO:0006928: cell motion 20 12.5 1.14E-06
 GO:0048870: cell motility 16 10 1.47E-06
 GO:0051674: localization of cell 16 10 1.47E-06
 GO:0016477: cell migration 15 9.375 2.16E-06
 GO:0001568: blood vessel 11 6.875 3.82E-04
development
Fig. 1 - Hierarchical clustering heatmaps of the differentially ex-  GO:0001944: vasculature 11 6.875 4.62E-04
pressed miRNAs. The gradient of colors from blue to orange repre- development
sents expression values from low to high. *represents the miRNA
expressed from the mature miRNA or the miRNA with relatively  GO:0048514: blood vessel 10 6.25 5.46E-04
low-abundance. morphogenesis
Pathway enrichment analysis
 hsa05210: Colorectal cancer 8 5 2.47E-04
Total 66 miRNAs were identified to be differentially expressed  hsa04520: Adherens junction 7 4.375 0.001001
between the short-OS and long-OS groups based on the cutoff
 hsa05220: Chronic myeloid leukemia 6 3.75 0.00528
value of FDR <0.05 and |log FC| >1.
After the expression values of the 66 differentially ex-  hsa04350: TGF-beta signaling 6 3.75 0.009825
pressed miRNAs were preprocessed, hierarchical clustering of pathway
63 miRNAs in 19 samples was performed. As shown in Figure 1,  hsa05212: Pancreatic cancer 5 3.125 0.023013
the samples from the short-OS group and long-OS group are  hsa05412: Arrhythmogenic right 5 3.125 0.027435
clearly distinguished based on the expression profiling of the ventricular
differentially expressed miRNAs. Meanwhile, the expression  hsa04512: ECM-receptor interaction 4 2.5 0.01338
patterns of miRNAs are clustered into 2 subclusters. In subclus-  hsa05410: Hypertrophic 4 2.5 0.013726
ter 1, 33 miRNAs show high expression levels in the short-OS cardiomyopathy
group and low expression levels in the long-OS group. A total  hsa05414: Dilated cardiomyopathy 4 2.5 0.016233
of 30 miRNAs show low expression levels in the short-OS group
and high expression levels in the long-OS group (subcluster 2). GO = gene ontology.

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Wang et al e229

Fig. 2 - Protein–protein interaction (PPI) network for miRNA target genes and some normal interactional proteins. The size of the nodes
represents the node degree in the network – the higher the degree, the bigger the node. The color of the nodes represent the centrality,
the higher the centrality, the deeper the color (red) and the lower the centrality, the lighter the color (green).

PPI network of target genes TABLE II - Top 10 gene nodes with high node degrees in the network
and the corresponding topology parameters
Based on the information from the HPRD database, we col-
lected the protein pairs involved with the 163 miRNA target Node Node degree Centrality ASPL CC
genes. A PPI network with 222 nodes and 562 edges was TGFBR1 49 0.421756 2.371041 0.058693
constructed using the Cytoscape software (Fig. 2). After
further analyzing the topological characteristics of the SMAD4 48 0.426641 2.343891 0.045404
PPI network, we obtained 10 hub nodes, including trans- SHC1 31 0.37585 2.660633 0.054187
forming growth factor, beta receptor 1 (TGFBR1), SMAD ACVR1 24 0.359935 2.778281 0.065217
family member 4 (SMAD4), Src homology 2 domain con-
TGFBR2 22 0.378425 2.642534 0.137255
taining transforming protein 1 (SHC1), activin A receptor
type I (ACVR1) and transforming growth factor beta recep- RET 20 0.350238 2.855204 0.1
tor II (TGFBR2). The topological characteristics for the 10 ITGB3 17 0.345313 2.895928 0.054945
significant nodes are listed in Table II. Then the biological CDKN1A 17 0.324047 3.085973 0
functions of the 10 genes were further analyzed with the
GeneMANIA tool. According to the annotation informa- EZR 16 0.325 3.076923 0.038462
tion recorded in GeneMANIA, a PPI network involved with BCL2 16 0.342636 2.918552 0
the 10 target genes and 20 normal human genes was es- AVE (mean 5.06306 0.28866 3.537157 0.119178
tablished (Fig. 3). GeneMANIA functional annotation for value)
the 10 hub genes showed that the 10 genes were mainly
involved in the pathway-restricted SMAD protein phosphor- ACVR1 = activin A receptor type I; ASPL = average shortest path length;
CC = clustering coefficient; SMAD4 = SMAD family member 4; SHC1 = Src ho-
ylation, the transforming growth factor beta receptor signal- mology 2 domain containing transforming protein 1; TGFBR1 = transforming
ing pathway and transforming growth factor beta binding growth factor, beta receptor 1; TGFBR2 = transforming growth factor, beta
(Tab. III). receptor II.

© 2015 Wichtig Publishing

e230 Chemotherapy resistance analysis

Multivariate statistical analysis

To verify the significance of the differentially expressed

Fig. 3 - A network was constructed from GeneMANIA annotation
information for 10 important genes, including physical interac-
tion, genetic interaction, co-expression and shared pathways
and protein structure domain. Black nodes represent 10 impor-
tant genes, and gray nodes represent gene interactions with
the black nodes. The network also contains 20 normal human
proteins.
miRNAs through a statistical analysis, we used 2 kinds of mul-
tivariate statistical methods to analyze these miRNAs.
A binary logistic regression model (27) was applied to cal-
culate the expression values of 66 significant miRNAs and the
correlations of the 2 classified groups of patients. As shown in
Table IV, 40 miRNAs were found to effectively distinguish the
short-OS and long-OS patients in the binary logistic regres-
sion model (p<0.05). A discriminant analysis model (28) was
also applied to analyze the significance of 66 miRNAs. Based
on the expression values of the 66 miRNAs in 19 patients,
8 miRNAs (i.e., hsa-miR-1914, hsa-miR-4281, hsa-miR-1274a,
hsa-miR-1249, hsa-miR-1207-3p, hsa-miR-466, hsa-miR-1290
and hsa-miR-31) were identified to be significant (p<0.05;
Tab. V), which was consistent with the results of the binary lo-
gistic regression model. Discriminant analysis suggested that
the 8 miRNAs could completely distinguish the short-OS and
long-OS patients (accuracy 100%).
From the miRecord, miRTarBase and Tarbase 6.0, we ob-
tained 26 target genes of the 8 significant miRNAs. Based on
the information from the HPRD, 19 target genes were mapped
into the PPI network, among which 7 target genes were found
to have high node degrees and were mainly involved in control
of cell fate decisions, E26 oncogene homolog 1 and enhancing
angiogenesis (Tab. VI).
Discussion

TABLE III - GeneMANIA functional annotation
Feature
Pathway-restricted SMAD protein
phosphorylation
Transforming growth factor beta
receptor signaling pathway
Cytokine receptor binding
Regulation of SMAD protein phos-
phorylation
Cellular response to transforming
growth factor beta stimulus
Response to transforming growth
factor beta stimulus
Transforming growth factor beta
binding
Regulation of pathway-restricted
SMAD protein phosphorylation
Transmembrane receptor protein
serine
Transforming growth factor beta
receptor binding
FDR = false discovery rate.
As the mortality rate for pancreatic cancer patients is al-
most 0.99 (29), there is an urgent need for understanding the
molecular mechanisms underlying this disease to develop
FDR Count Total
better detection, diagnostic and prognostic markers as well
1.54E-09 7 36 as therapeutic targets that could improve clinical manage-
ment and therapeutic outcomes for these patients. In this
study, we downloaded the miRNA expression profiles from
3.48E-09 9 141 PDAC patients with similar clinicopathological characteristics
and chemotherapy but different clinical outcomes (short-OS
3.48E-09 9 139 vs. long-OS groups). Poor prognosis had an association with
chemotherapy resistance (30). The miRNAs that showed dif-
5.50E-09 6 25 ferent expression levels between the short-OS and long-OS
groups were selected. The relevant miRNA target genes were
collected, and the functions for significant genes were further
5.50E-09 9 160
investigated. The purpose of this paper was to explore the
chemotherapy sensitivity–related mechanism and the poten-
5.50E-09 9 160 tial biomarkers for prognosis prediction.
Our results showed that 66 miRNAs were differentially
expressed, and hierarchical cluster analysis for these miRNAs
1.34E-08 5 12 showed a good separation of the samples from the short-OS
and long-OS groups. A total of 163 target genes for the dif-
1.69E-08 6 32 ferentially expressed miRNAs were collected. Pathway en-
richment analysis for the 163 miRNA target genes revealed
that the significant pathways were mainly involved with
4.45E-08 9 211 cancer development and heart function such as arrhythmo-
genic right ventricular, hypertrophic cardiomyopathy and
5.16E-08 5 16 dilated cardiomyopathy. As outlined in previous reports, the
cardiovascular disease can lead to various pathophysiologi-
cal changes which may affect pharmacokinetics such as drug
absorption, distribution, metabolism and excretion (31). The

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Wang et al e231

TABLE IV - MiRNAs identified by binary logistic regression TABLE V - MiRNAs identified by discriminant analysis

miRNA Score df p Value miRNA Short OS Long OS

hsa-miR-1197 16.548 1 0.000 hsa-miR-1914 -2.352 -8.595
hsa-miR-1914 14.674 1 0.000 hsa-miR-4281 0.194 0.685
hsa-miR-4321 11.989 1 0.001 hsa-miR-1274a -0.211 -0.802
hsa-miR-940 11.791 1 0.001 hsa-miR-1249 -2.940 -10.798
hsa-miR-4281 11.670 1 0.001 hsa-miR-1207-3p 11.548 34.288
hsa-miR-1207-3p 11.556 1 0.001 hsa-miR-466 20.182 69.342
hsa-miR-1914 11.279 1 0.001 hsa-miR-1290 -3.835 -12.264
hsa-miR-1246 11.157 1 0.001 hsa-miR-31 0.214 0.760
hsa-miR-371-5p 10.821 1 0.001 (Constant) -198.728 -1,710.581
hsa-miR-181a-2 10.469 1 0.001 OS = overall survival.
hsa-miR-887 9.840 1 0.002
hsa-miR-3610 9.747 1 0.002 TABLE VI - F unctional annotations of 7 important genes regulated
hsa-miR-532-5p 9.591 1 0.002 by 8 miRNAs identified by discriminant analysis
hsa-miR-483-3p 9.520 1 0.002
Gene Node degree Function annotation
hsa-miR-1249 9.348 1 0.002 symbol
hsa-miR-92a-2 9.048 1 0.003 RET 20 Ret proto-oncogene
hsa-miR-25 9.028 1 0.003
RHOA 14 Ras homolog gene family
hsa-miR-466 8.633 1 0.003
ETS1 11 E26 oncogene homolog 1
hsa-miR-1915 8.321 1 0.004
NUMB 9 Control of cell fate decisions
hsa-miR-1200 8.280 1 0.004
hsa-miR-1274a 8.270 1 0.004 TIAM1 7 T-cell lymphoma invasion and metastasis 1

hsa-miR-149 7.781 1 0.005 ITGA5 6 Enhance angiogenesis

hsa-miR-619 7.756 1 0.005 YY1 6 Multifunctional transcription factor
hsa-miR-4284 7.528 1 0.006
hsa-miR-1911 6.723 1 0.010
cardiac dysfunctions may result in hypoperfusion to the drug
hsa-miR-4286 6.679 1 0.010 clearance site, a decline in the volume of drug distributed and
hsa-miR-1290 6.668 1 0.010 impairment of drug clearance (32). The limited distribution
of anticancer drug in tumor tissue is related to the resistance
hsa-miR-3681 6.643 1 0.010 of cancers to chemotherapy (33). In addition, compelling evi-
hsa-miR-3196 6.632 1 0.010 dence shows that the arachidonic acid (AA) cascade, which is
important in cardiovascular disease, is involved in the devel-
hsa-miR-4271 6.606 1 0.010
opment of pancreatic adenocarcinomas (34). The treatment
hsa-miR-197 6.213 1 0.013 and prevention of cardiovascular disease with therapies such
hsa-miR-1236 6.165 1 0.013 as β-blockers, inhibitors of AA-metabolizing enzymes and a
low-fat diet for cardiovascular disease are also effective for
hsa-miR-671-3p 6.086 1 0.014 PDAC. Although there is no direct association between heart
hsa-miR-326 5.456 1 0.020 disease and PDAC, heart functions may be related to drug re-
sistance and may influence the survival of PDAC patients.
hsa-miR-31 4.981 1 0.026
In addition, the target genes of differentially expressed
hsa-miR-934 4.815 1 0.028 miRNAs in PDAC patients were mapped into a PPI network.
hsa-miR-412 4.329 1 0.037 Ten genes were identified to be the hub nodes, including
TGFBR1, TGFBR2, SMAD4, ACVR1 and SHC1. TGFBR1 and
hsa-miR-4251 4.003 1 0.045 TGFBR2 encode transforming growth factor, beta receptor
hsa-miR-204 3.952 1 0.047 I and transforming growth factor, beta receptor II, respec-
tively, which are members of the transforming growth fac-
hsa-miR-4310 3.946 1 0.047 tor beta (TGFB) receptor subfamily. TGFBR1 and TGFBR2

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are TGF-beta receptors and play roles in transducing the
TGF-beta signal. TGF-beta modulates many aspects of cel-
lular function, and the disruption of the activity of TGFB
super members (such as TGFBR1 and TGFBR2) contributes
to a variety of diseases such as various cancers and pul-
monary hypertension (35, 36). SMAD family member 4
(SMAD4), encoded by the SMAD4 gene, is a member of the
Smad signal transduction protein family. Smad proteins are
phosphorylated and activated by transmembrane serine-
threonine receptor kinases in response to TGF-beta signal-
ing (37). Previous evidence has implied that the mutation of
the SMAD4 gene is correlated with the outcomes of patients
with pancreatic cancer (38). SMAD4 inactivation has close
associations with shorter OS in pancreatic cancer patients
with resected surgeries (39). ACVR1 encodes the Activin A
receptor type I, which is a signaling protein belonging to the
TGF beta superfamily. Mutations of ACVR1 have been found
in patients with pancreatic cancer (40). Src homology 2 do-
main containing transforming protein 1 (SHC1), encoded by
SHC1, is reported to have functions in regulation of apop-
tosis and drug resistance in mammalian cells (41). Recent
evidence shows that SHC1 regulated by mi-365 is associat-
ed with gemcitabine resistance in patients with pancreatic
cancer (42). Therefore, the significant gene nodes in the PPI
network may play key roles in the mechanism of chemo-
therapy resistance and may be potential genetic biomarkers
for the prognosis of patients with pancreatic cancer. How-
ever, a large number of further studies are warranted.
In addition, by multivariate statistical methods, 8 of 66
differentially expressed miRNAs were found to be the most
significant and could completely distinguish the samples from
the 2 groups (short-OS and long-OS). Seven target genes of
the 8 significant miRNAs were proven to have higher node de-
grees in the PPI network (i.e., RET, ETS1, RHOA, NUMB, TIAM,
ITGA5 and YY1).
RET and ETS1 are important proto-oncogenes associated
with tumorigenesis (43, 44). RHOA and NUMB are involved
in the important process of cell cycle and regulate cell pro-
liferation and apoptosis (45, 46). TIAM1 and ITGA5 regulate
invasion and metastasis of immune cells and angiogenesis,
which are closely associated with tumorigenesis, metasta-
sis and chemotherapy resistance (47, 48). YY1 is a kind of
multifunctional transcription factor controlling complex bio-
logical processes (49). The 7 important genes regulated by 8
miRNAs affect the biological processes of cell cycle, immune
system and angiogenesis, and drug resistance of pancreatic
cancer. So the 8 significant miRNAs (hsa-miR-1914, hsa-
miR-4281, hsa-miR-1274a, hsa-miR-1249, hsa-miR-1207-3p,
hsa-miR-466, hsa-miR-1290 and hsa-miR-31) may play key
roles in regulating the resistance of patients with pancreatic
cancer.
In conclusion, the differentially expressed miRNAs be-
tween PDAC patients with short-OS and long-OS affected
their resistance by regulating their target genes. Heart
function related pathway, cell cycle, immune system and
angiogenesis may be dysregulated in patients with poorer
prognosis. The significant nodes (such as TGFBR1, TGFBR2,
ACVR1 and SHC1) in the PPI network may be potential mark-
ers for predicted the outcomes of patients with pancreatic
cancer. Although some significant genes and miRNAs were
Chemotherapy resistance analysis
found to be related to prognosis of pancreatic cancer, lack
of the experimental evidence was a limitation of our work.
Our findings may prompt further prospective studies and re-
search into the predicted and therapeutic role of the signifi-
cant genes and differentially expressed miRNAs. However,
extensive further studies should be conducted to verify the
findings of our work.
Disclosures
Financial support: No grants or funding have been received for this
study.
Conflict of interest: All authors declare that they have no conflict of
interest to state.
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