Computational Inferring of Risk
Subpathways Mediated by Dysfunctional
Non-coding RNAs
Yanjun Xu, Yunpeng Zhang, and Xia Li
Abstract
Non-coding RNAs mediated core elements of
pathways contributes to the disorder of
biological function in diseases. Identification
of non-coding RNAs mediated subpathways
not only can help for deciphering the patho-
genic mechanism of complex diseases, but
also can gain insight into the functional roles
of non-coding RNAs in human diseases. Here,
we summarized the general steps for
identifying non-coding RNA mediated
subpathways and overviewed two of our pre-
viously developed methods, Subpathway-
GMir and Subpathway-LNCE, which were
designed to identify miRNAs and lncRNAs
mediated risk subpathways respectively. We
identified the key subpathway regions by
integrating non-coding RNA-target gene
associations, interesting genes and
non-coding RNAs and pathway topologies.
By applying methods to several disease
datasets, we confirmed that our methods is
effective in identifying risk subpathways and
also can help uncover key non-coding RNAs
in diseases. Additionally, reproducibility and
robustness analysis demonstrated our methods
are reliable.
Y. Xu · Y. Zhang · X. Li (*)
College of Bioinformatics Science and Technology,
Harbin Medical University, Harbin, Heilongjiang, China
e-mail: zhangyp@hrbmu.edu.cn; lixia@hrbmu.edu.cn
# Springer Nature Singapore Pte Ltd. 2018
X. Li et al. (eds.), Non-coding RNAs in Complex Diseases, Advances in Experimental Medicine and
Biology 1094, https://doi.org/10.1007/978-981-13-0719-5_9
9
Keywords
Non-coding RNA · Subpathway · Pathway
topology · Integration · Network
9.1 Introduction
Recently, non-coding RNAs have received
increased attention with the development of
whole genome and transcriptome sequencing
technologies [1, 2]. A large number of studies
have demonstrated the important roles of
non-coding RNAs in human diseases.
MicroRNAs mediated the transcription processes
and thus can impact the initiation, progression,
and prognosis of tumors [3–6]. LncRNAs also
have be confirmed as key regulators of diverse
cellular processes [7–9], such as genomic
imprinting [10], cell differentiation [11] and
immune responses [12]. Especially, lncRNAs
can regulate the expression of mRNAs by com-
peting common miRNA binding sites with
mRNAs [13, 14]. PTEN, a critical tumor suppres-
sor gene, has been successfully validated that it
can act as ceRNA of PTENP1 which is a PTEN
pseudogene [15, 16]. H19 is an oncogene which
has been shown as a ceRNA to adsorb miR-138
and miR-200a and then impact the core gene of
mesenchymal cells (e.g., ZEB1/ZEB2) in colo-
rectal tumor [17]. As non-coding RNAs play
such critical roles in biological system, it is essen-
tial to develop methods which is helpful for
87
88
revealing the action mechanism and functional
roles of non-coding RNAs and enhance the
understanding of etiology in human diseases.
Recently, a series of databases and methods
have been provided. TarBase [18], miR2Disease
[19], mirTarBase [20] and miRecords [21] are
databases that aim to collect experimentally
validated associations between miRNAs and tar-
get genes. Naamati et al. provided method to
investigate the coordinated action of miRNAs to
impact biological pathways [22]. Guttman et al.
inferred the putative functions for lincRNAs
based on their co-expressed mRNAs
[1]. LncRNA2Function web tool annotated the
function of lncRNAs based on the expression
correlation of lncRNA and mRNA, which is
extracted from the RNA-Seq data for 19 normal
tissues [23]. These databases and methods are all
useful for investigating the function of
non-coding RNAs. However, our currently
knowledge relevant with the functional roles and
regulation mechanism of non-coding RNAs con-
tribute to disease pathologies is still limit.
Identifying non-coding RNAs mediated
biological pathways is an effective strategy to
address this challenge. Moreover, it have been
demonstrated that abnormalities in “subpathway
regions” may be more relevant with the disease
etiology comparing with entire pathways
[24, 25]. Thus, it is reasonable to focus on
Fig. 9.1 The pipeline overview of identifying dysregulated subpathways mediated by non-coding RNAs
Y. Xu et al.
identifying key dysfunctional sub-regions
mediated by non-coding RNAs in diseases.
Here, we introduced two of our previously
developed methods including Subpathway-GMir
[26] and Subpathway-LNCE [27], both of which
aim to identify dysregulated subpathways
mediated by non-coding RNAs. Specifically,
Subpathway-GMir was designed for identifying
miRNA mediated risk subpathways by consider-
ing interesting genes, miRNAs and pathway
topologies; while Subpathway-LNCE aims to
locate key subpathway regions that competitively
regulated by lncRNAs in diseases. These two
methods are not only helpful for deciphering the
functional roles of lncRNAs, but also can gain
insight into the underlying mechanism.
9.2 Methods
The general steps of non-coding RNA mediated
subpathway identification processes is depicted in
Fig. 9.1. It contains four main components:
(I) Extraction of pathway topology;
(II) Reconstructing pathways that involved
non-coding RNAs by integrating non-coding
RNA-gene association data and pathway
topologies; (III) Locating candidate key
subpathway regions based on interesting genes,
non-coding RNAs and/or molecular profiles, etc.;
9 Identification of Risk Subpathways
(IV) Evaluating the significance of candidate
subpathways. The details of the processes are
described below.
9.2.1 Extraction of Pathway
Topology
We converted pathway structures of KEGG into
undirected graphs with nodes represent genes and
edges represent interactions between genes by the
use of “iSubpathwayMiner”, an R package devel-
oped by us previously [24, 25]. In details, the
corresponding XML file of pathways were
downloaded from the KEGG KGML (http://
www.genome.jp/kegg/) which represent the path-
way information, firstly. Then, the pathway struc-
ture (gene-gene interaction information) were
extracted from these KGML files. For each meta-
bolic pathway, the relationship of enzymes were
extracted from each XML file. Two enzymes are
connected by an edge if their corresponding
reactions have a common compound. The meta-
bolic pathways were simplified to an undirected
graph with enzymes (genes) as nodes. Two genes
are connected by an edge if they share a common
compound in the corresponding reactions. For
each of other pathways (e.g. signaling pathways),
two genes are connected with each other if their
annotated proteins were interacted in the original
KEGG pathway map. Finally, the gene-gene
interaction (pathway structure) were extracted
for each pathway as an undirected graph.
9.2.2 Reconstruction of Pathway
Topology Embedded with Non-
coding RNAs
In this section, the non-coding RNAs were
embedded into each pathways based on the
non-coding RNA-gene associations. Briefly, for
each pathway, non-coding RNAs with at least one
associated target were embedded as non-coding
RNA node into the pathway by connecting it to its
verified targets contained in the corresponding
pathway. Finally, we obtained the reconstructed
KEGG pathways which contain non-coding RNA
89
nodes and non-coding RNA-gene interaction
edges. For example, in Subpathway-GMir
method, the experimentally verified miRNA-
target interactions were used for reconstructing
KEGG pathway structures. These miRNA-target
interactions were integrated from miRTarBase
[20], mir2Disease [19], miRecords (V4.0) [21]
and TarBase (V6.0) [18] databases. In this
method, the reconstructed KEGG pathways con-
tain miRNA nodes and miRNA-gene interaction
edges. In the Subpathway-LNCE method, the
lncRNA-gene associations were constructed
based on the competing endogenous RNA
hypothesis [28]. Firstly, the lncRNA-miRNA-
mRNA competing relationships were
constructed. We collected the miRNA and its
target mRNA from four public databases
(TarBase, mirTarBase, mir2Disease, miRecords
(V4.0)), and lncRNA-miRNA interactions were
obtained from our previously work and StarBase
[29]. For each lncRNA, we recognize its compet-
itive regulated mRNA(s) based on their shared
miRNAs, meeting the following two conditions.
(i) hypergeometric test p-value of shared miRNAs
under a threshold 0.05 (ii) Jaccard Coefficient of
shared miRNAs rank at top 20%. Then, we
mapped lncRNAs into pathway graphs as nodes
by linking to their regulated-mRNAs, whose
Pearson Correlation Coefficient had reached a
significant threshold (p < 0.05). Finally, we
obtained condition-specific lncRNA competi-
tively regulated signal pathways (LRSP), which
included lncRNA nodes and lncRNA-mRNA
competitively regulated edges.
9.2.3 Locating Dysfunctional
Subpathway Regions Mediated
by Non-coding RNAs
Then, the dysfunctional subpathway regions were
located within these reconstructed pathways
based on interesting nodes (genes and/or
non-coding RNAs) or molecule profiles. In
Subpathway-GMir [26] and SubpathwayLNCE
[27] method, we used “lenient distance” similar-
ity combined with network topology structure
[25] to locate the dysfunctional subpathway
90
regions based on interesting nodes. Briefly, we
first mapped the interesting nodes into the above
reconstructed pathways which were embedded
with non-coding RNAs. For each signature node
pairs, we extracted the shortest path between
them. If the number of non-interesting molecules
in the shortest path of each signature pairs was no
longer than n, we merge the nodes which among
the shortest path between any two signature nodes
into modules. While, the number of nodes in the
molecule sets within pathway no less than s were
regarded as candidate subpahtways regions that
mediated by non-coding RNAs. Here, the param-
eter n and s control the intensity of regulated
signal and the size of the candidate subpathways,
respectively.
9.2.4 Calculating Significance
of Subpathway Regions
After located the subpathway regions within each
entire pathway, some strategies were used to eval-
uate the significance of each candidate
subpathways. Some mostly used statistical
methods including hypergeometric test, Fisher
Exact test, etc. Furthermore, the random pertur-
bation strategy was also one of the mostly used
method for evaluating the significance of candi-
date subpathways. In the Subpathway-GMir
method [26], the following formula based on
hypergeometric method was used to evaluate the
statistic significance of the difference between
two study conditions for located subpathways.
 
t g þt mir 1
r g þr mir mg þmmir r g r mir
X k ng þnmir k
P¼1  
mgþmmir ng þnmir ngþnmir
k¼0 ngþnmir
Where mg (mmir) is the number of genes
(miRNAs) in entire genome (miRNAome), and
ng (nmir) is the number of all differentially
expressed genes (miRNAs). For single
subpathway containing rg genes (rmir miRNAs),
there are tg (tmir) genes (miRNAs) participated
in it.
Y. Xu et al.
In Subpathway-LNCE method [27], We used
the Wallenius approximation methods to estimate
whether the candidate subpathway was signifi-
cantly competing regulated by lncRNAs compar-
ing with random. The following parameters were
required: (i) the number of interesting genes (x);
(ii) the number of background genes (n); (iii) the
number of background genes annotated in
subpathway (m1); (iv) the number of interesting
genes involved in subpathway (m2); (v) the
weight of subpathway (w), which was computed
as the following formula, suggested the intensity
of lncRNAs regulated for the corresponding
subpathway. The parameter Gp and Gl represent
the number of genes and genes that competitively
regulated by lncRNAs in subpathway, respec-
tively. In the Subpathway-LNCE method, we set
β ¼ 1. The Wallenius approximation method was
performed using R package BiasedUrn [30].
  
Gl
W ¼ 1 þ β log2 ð9:2Þ
Gp
9.3 Case study
9.3.1 Identifying Metabolic
Subpathways Mediated by
miRNAs in LIHC
We used Subpathway-GMir method to identify
liver hepatocellular carcinoma (LIHC) associated
subpathways that mediated by miRNAs. First, we
identified risk genes and miRNAs of LIHC based

on the gene and miRNA expression profile from
ð9:1Þ TCGA database (https://cancergenome.nih.gov/).
The miRNA expression profile contains
100 tumor and 50 normal samples; while
17 tumor and 9 normal samples were involved
in the gene expression profile. We obtained 3357
differential genes and 394 differential miRNAs,
which regarded as risk genes/miRNAs, by com-
bining the fold change and edgeR methods. Then,
we applied Subpathway-GMir method to identify
miRNA mediated risk subpathways for LIHC.
The results shown that there were 29 subpathways
9 Identification of Risk Subpathways
Fig. 9.2 MiRNA mediated risk subpathway region of
arachidonic acid metabolism in LIHC. Rectangle, circle
and triangle nodes represent protein coding genes,
that belonged to 29 distinct complete pathways at
FDR <0.01, 18 of which have been reported to be
related with cancers. For example, the
path:00590_1 (Fig. 9.2) shows the most signifi-
cance and takes an important part in arachidonic
acid metabolic (AAM) pathway which can sup-
press the development and apoptosis of
hepatomas in light of many studies [31, 32]. The
path:00590_1 contains ten genes and three
miRNAs, including Cyclooxygenase-2 (COX-2),
Prostaglandin H2 (PGH2) and miR-101, etc. In
vitro and animal models experiments
demonstrated that overexpression of COX-2
which is a differential gene in this LIHC dataset
can promote LIHC cell growth [33]. Moreover,
miR-101 inhibits COX-2 expression and its
downstream metabolite PGH2 to promote pro-
gression of multiple cancers [34, 35]. These
findings indicate that Subpathway-GMir can
effectively identify key sub-regions that are rele-
vant with study condition.
9.3.2 Identifying Signal Subpathways
Competitively Regulated by
LncRNAs for Prostate Cancer
We used Subpathway-LNCE method success-
fully identified dysregulated subpathways that
competitively regulated by lncRNAs in prostate
cancer. The matched lncRNA and mRNA
91
miRNAs and metabolites, respectively. Red nodes repre-
sent differential genes/miRNAs in the corresponding
LIHC dataset
expression profiles for prostate cancer were
extracted from SRP002628 which stored in SRA
database [36]. Most of these dysregulated
subpathways were associated with the initiation
and development of tumor. Specifically, path:
04510_1 is an important sub-region within focal
adhesion pathway (Fig. 9.3). In this subpathway,
extracellular matrix (ECM) was an upstream pro-
tein, which has been reported to implicate with
the metastasis of tumors [37, 38]. Interestingly,
this protein coding gene was coordinately
regulated by four lncRNAs (Fig. 9.3), in particu-
lar, MEG8 and AC078937.4.1 were differentially
expressed in the prostate cancer dataset. Another
dysregulated lncRNA, LINC00087, which com-
petitively regulated two critical cancer genes
including VEGF and CCND2. This indicates
that LINC00087 may play an important role in
prostate cancer. Furthermore, DLEU2 and
HOTAIRM1 were also competitively regulated
this subpathway, which had been reported play
important role in the leukemia cells
[39, 40]. DLEU2 was the host gene of miR-15a
and miR-16-1 which are important in
tumorigenesis [41]. In summary, these results
shown that Subpathway-LNCE method can iden-
tify lncRNAs competitively regulated
subpathways and potential key lncRNA underly-
ing disease condition.
92 Y. Xu et al.

Fig. 9.3 LncRNA

competitively regulated
subpathway region of Focal
adhesion pathway in
prostate cancer. Triangle
and circle nodes represent
lncRNAs and protein
coding genes, respectively.
Red node label represent
differential genes/lncRNAs
in the corresponding
prostate cancer dataset

9.4 Evaluation of Method 9.4.2 Reproducibility and Robustness

Analysis
9.4.1 Comparison with Other
Methods 9.4.2.1 Reproducibility Analysis
As non-coding RNA/gene expression profiles that
from different experimental platforms exist tech-
After developed the method, an important step is
nical difference, a reliable method should be
to evaluate and demonstrate the superiority of
resistant for this. Reproducibility analysis aims
it. Comparison with previous methods is one of
to evaluate whether pathway identification
the mostly used strategies. For example, we com-
method could have a high concordance when
pared Subpathway-GMir method with the ORA
applied to datasets from different platforms but
[42] and the Kretschmann et al. method [43, 44]
which is underlying the same study condition.
based on the LIHC dataset. Specifically, the ORA
Both Subpathway-GMir and Subpathway-LNCE
method only focuses on genes while the
methods were all have a relative high degree of
Kretschmann et al. method only focuses on
reproducibility. In order to evaluate the reproduc-
miRNA. We found that pathways identified by
ibility of Subpathway-GMir method, we collected
these two previous methods were all included in
another LIHC related dataset which was
the pathway list that identified by Subpathway-
downloaded from GEO database [45] and
GMir method. And many pathways that closely
provided by a different research group. The
related with the initiation and progression of
dataset accession number for miRNA and gene
tumor were missed by these two methods
is GSE36915 and GSE46408, respectively. Then,
(Table 9.1). This indicates that Subpathway-
we applied Subpathway-GMir to this dataset. As
GMir is more effective to detect LIHC-relevant
a result, up to 83% pathways that identified based
pathways than other methods.
the second LIHC dataset were also identified in
the original LIHC dataset; while these two LIHC
9 Identification of Risk Subpathways 93

Table 9.1 Comparison of subpathway-GMir with other methods

Pathway name Subpathway-GMir ORA Kretschmann et al.
Glycolysis/Gluconeogenesis √ √
Purine metabolism √ √
Pyrimidine metabolism √ √
Inositol phosphate metabolism √ √
Glycerophospholipid metabolism √ √
Metabolism of xenobiotics by cytochrome P450 √ √
Lysine degradation √ √
Arachidonic acid metabolism √
Fatty acid metabolism √ √
Glycerolipid metabolism √
Retinol metabolism √
Fructose and mannose metabolism √
Drug metabolism – cytochrome P450 √
Sphingolipid metabolism √
Pentose phosphate pathway √
Steroid hormone biosynthesis √
Tryptophan metabolism √
Starch and sucrose metabolism √
Arginine and proline metabolism √ √
Ether lipid metabolism √
Alanine, aspartate and glutamate metabolism √
Pyruvate metabolism √
beta-Alanine metabolism √
Tyrosine metabolism √
Valine, leucine and isoleucine degradation √
Glutathione metabolism √
Glycine, serine and threonine metabolism √
Galactose metabolism √
Histidine metabolism √
Note: pathway name marked with bold represent it has been reported to associate with cancer. ‘√’ represents pathway
was identified by the corresponding method

related datasets only shared 30% of the differen-
tial genes and 5% of the differential miRNAs.
This indicates that the high reproducibility of
Subpathway-GMir was not due to common dif-
ferential genes and miRNAs. Similarly, we also
evaluate the reproducibility of Subpathway-
LNCE method based on three independent pros-
tate cancer datasets which were obtained from
SRA,TCGA and GEO databases, respectively.
The result demonstrated that Subpathway-LNCE
also has a high reproducibility.
9.4.2.2 Robustness Analysis
Robustness analysis is to evaluate whether
method is stable for the disturbance from
biological data that used in method. Disturb
factors that critical for method is a useful strategy
to evaluate the stability of method. For example,
the noise from expression profiles and false posi-
tive interactions within pathway topology may
impact the performance of both Subpathway-
GMir and Subpathway-LNCE methods. Thus, in
the evaluation process of these two methods, we
randomly deleted different percentage (5%, 10%,
. . ., 30%) of genes/non-coding RNAs from
expression profiles and different percentage
(5%, 10%, . . ., 30%) of edges within each path-
way, respectively. The results shown that our
method have a relative strong robustness in dis-
turbance of molecular profiles and pathway
topology.
94
9.5 Concluding Remarks
We summarized two our previously developed
methods (Subpathway-GMir and Subpathway-
LNCE) that devoted to identify non-coding
RNA mediated dysregulated subpathways for
complex diseases. In our methods, we focused
on identifying the key sub-regions within entire
pathway, which is consistent with the concept that
key local sub-regions, but not the entire
pathways, were dysregulated in diseases. More-
over, we incorporated pathway topologies and
non-coding RNA-target gene interaction data
and thus these two methods could provide the
detail interaction information between genes and
non-coding RNAs that associated with the disor-
dered subpathway. In summary, our methods will
not only be helpful for understanding the mecha-
nism of complex diseases, but also will greatly
facilitate the exploring of non-coding RNA func-
tion and their roles in human diseases. Especially,
it is more precision to characterize the functional
roles of non-coding RNAs at subpathway level.
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