Mol Neurobiol

DOI 10.1007/s12035-016-0353-x

Genome-Wide DNA Methylation Patterns Analysis of Noncoding

RNAs in Temporal Lobe Epilepsy Patients
Wenbiao Xiao 1 & Yuze Cao 2,3 & Hongyu Long 1 & Zhaohui Luo 1 & Shuyu Li 1 & Na Deng 1 &
Jianjian Wang 2 & Xiaoyan Lu 2 & Tianfeng Wang 2 & Shangwei Ning 4 & Lihua Wang 2 &
Bo Xiao 1

Received: 13 August 2016 / Accepted: 13 December 2016

# Springer Science+Business Media New York 2017

Abstract Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy and frequently evolving drug
resistance. Although there is growing consensus that noncoding ribonucleic acids (ncRNAs) are modulators of TLE,
the knowledge about the deoxyribonucleic acid (DNA)
methylation patterns of ncRNAs in TLE remains limited.
In the current study, we constructed DNA methylation profiles from 30 TLE patients and 30 healthy controls for
ncRNAs, primarily focusing on long ncRNAs (lncRNAs)
and microRNAs (miRNAs), by reannotating data of DNA
methylation BeadChip. Statistics analyses have revealed a
global hypermethylation pattern in miRNA and lncRNA
gene in TLE patients. Bioinformatic analyses have found

Wenbiao Xiao and Yuze Cao contributed equally to this work.

Electronic supplementary material The online version of this article
(doi:10.1007/s12035-016-0353-x) contains supplementary material,
which is available to authorized users.
* Shangwei Ning
ningsw@ems.hrbmu.edu.cn
* Lihua Wang
wanglh211@163.com
* Bo Xiao
mdboxiao@163.com
1

Department of Neurology, Xiangya Hospital, Central South

University, Changsha 410008, China

Department of Neurology, The Second Affiliated Hospital, Harbin

Medical University, Harbin 150081, China

Department of Neurology, Peking Union Medical College Hospital,

Beijing 100032, China

College of Bioinformatics Science and Technology, Harbin Medical

University, Harbin 150081, China

aberrantly methylated miRNAs and lncRNAs are related to

ion channel activity, drug metabolism, mitogen-activated
p r o t e i n k i n a s e ( M A P K ) s i g n a l i n g p a t h w a y, a n d
neurotrophin signaling pathway. Aberrantly methylated
ncRNA and pathway target might be involved in TLE development and progression. The methylated and
demethylated ncRNAs identified in this study provide novel insights for developing TLE biomarkers and potential
therapeutic targets.
Keywords Temporal lobe epilepsy (TLE) . DNA
methylation . Long noncoding RNA (lncRNA) . MicroRNA
(miRNA) . Epigenetic

Introduction
Epilepsy is a group of neurological disorders characterized by recurrent, unprovoked epileptic seizures [1].
About 1 % of people worldwide have epilepsy, making
it one of the most common neurological diseases globally
[2, 3]. Temporal lobe epilepsy (TLE) is the most common
form of adult localization-related epilepsy [4], which is of
particular interest due to their progressive etiopathology
and frequently evolving drug resistance [5]. While great
progress has been made in the past decades in understanding of the neuropathology of TLE [6], the underlying
pathogenic and pharmacoresistance mechanisms of this
disorder remain poorly understood [7, 8].
MicroRNAs (miRNAs) are endogenous small noncoding RNAs (ncRNAs) (approximately 22 nucleotides) that
regulate post-transcriptional gene expression by translational repression and/or mRNA deadenylation/decay,
mainly through sequence-specific binding within the 3
UTR of mRNA transcripts [9, 10]. Recently, a series of

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studies have indicated that levels of over 100 different

miRNAs may either increase or decrease in experimental
and human epilepsy [11]. Dysregulation of miRNA involves the process of epileptogenesis, as well as maintenance and progression of the epileptic state [12]. What is
more, modulating individual miRNA can mitigate the attendant pathological features of TLE [13]. Long ncRNAs
(lncRNAs), defined as transcripts of >200 nucleotides
without protein-coding capacity [14], have been attracting
immense research interest. However, the field is still very
young that only a handful of lncRNAs have been characterized thoroughly [15]. LncRNAs play important roles in
brain development, neuronal function and maintenance of
synaptic plasticity, cognitive function, and memory. Also,
lncRNAs have been implicated in a variety of neurological disorders including epilepsy [16, 17]. For instance,
lncRNA Evf2 mouse mutants had reduced numbers of
GABAergic interneurons in early postnatal hippocampus
as well as synaptic inhibition in the adult hippocampus,
which may be implicated in epilepsy [18].
Deoxyribonucleic acid (DNA) methylation at gene promoters is crucial for gene repression [19]. Recent studies
have demonstrated that epigenetic mechanisms, including
DNA methylation, not only regulate the expression of
protein-encoding genes but also affect miRNAs and
lncRNAs [20, 21]. For example, maternally expressed
gene 3 (MEG3) is a lncRNA serving as a tumor suppressor gene; its promoter hypermethylation leads to a loss of
MEG3 expression in tumors [22]. In breast cancer research, Li et al. observed wide methylation changes more
frequently in the promoters of ncRNA genes than those of
protein-coding genes [21]. What is more, by comparative
analysis of expression and promoter methylation, MillerDelaney et al. identified a panel of differential methylation miRNAs and lncRNAs in TLE and proposed that
ncRNAs such as miRNA may be particularly sensitive
to DNA methylation [23]. However, little is known about
the regulatory mechanisms of miRNAs and lncRNAs in
TLE, especially on regulation by DNA methylation.
Whether miRNA and lncRNA methylation changes persist in and are important in TLE has not been explored.
Here, we used the Infinium Human Methylation 450K
BeadChip to quantitatively analyze methylation on over
485577 CpG sites in peripheral blood DNAs from TLE
patients and healthy controls. Subsequently, we conducted
a computational strategy to reannotate the DNA methylation arrays and obtained DNA methylation level of
miRNA and lncRNA promoters. Our analysis also led to
the functional enrichment analysis of differentially methylated miRNAs and lncRNAs. This study may help to
understand mechanisms regulating ncRNA gene expression and epileptogenesis and pharmacoresistance mechanisms in TLE.

Materials and Methods

Subjects
Peripheral blood samples were collected from a total of 30
patients (18 males and 12 females) and 30 sex- and agematched healthy controls. All patients met the criteria for the
diagnosis of TLE according to the International League
Against Epilepsy (ILAE). Drug-resistant epilepsy was defined
according to the global consensus definition developed by
ILAE [24]. All the patients were recruited from the
Department of Neurology at Xiangya Hospital from October
2010 to October 2014, and all patients went through comprehensive clinical examination, including medical history, physical and psychiatric examination, cranial magnetic resonance
imaging (MRI) scans, and electroencephalogram (EEG). The
demographic, clinical, brain MRI, and EEG profiles of the
subjects are summarized in Table 1 and Supplementary
Fig. S1. An informed consent to participate in this study
was obtained from each subject, and the study protocol was
approved by the Ethics Committee of Central South
University Xiangya School of Medicine and Xiangya
Hospital.
Whole Blood DNA Extraction and Genome-Wide DNA
Methylation Assays
Blood was collected from forearm vein in an EDTA tube and
then stored at 80 C. Whole blood DNA was isolated using a
commercial kit (Beijing Adly Biological Company, Beijing,
China) according to manufacturers instruction. DNA concentration and purity were determined in a NanoDrop spectrophotometer by measuring the optic density ratio of maximal absorbent wavelengths at 260 to 280 nm (OD260/280) using
salmon sperm DNA as an internal standard.
For genome-wide analysis, the Illumina Infinium Human
Methylation 450K BeadChip (Illumina, Inc., San Diego, CA,
USA) was used to interrogate DNA methylation at 485577
CpG sites, spanning 99 % of RefSeq genes. DNA samples
were sodium bisulfite converted and hybridized to arrays according to Illumina recommended protocols. Methylation at
individual CpG was reported as a methylation value, which
falls on a continuous linear scale ranging from 0
(unmethylated) to 1 (completely methylated).
MiRNA Annotation Files, miRNA Family, and miRNA
Cluster
Annotation files for human miRNAs were downloaded from
miRBase (http://www.mirbase.org/) [25]. We extracted the
annotated miRNA gene type from the Ensembl database
(http://asia.ensembl.org/index.html) [26]. MiRNA family is a
group of miRNAs that derive from the common ancestor.

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Table 1 Clinical characteristics
of individuals

TLE
No.

Control

30

30

Male (n = 18)

Age (mean SD)

25.28 11.75

28.17 13.96

Female (n = 12)
Inter-ictal EEG

Age (mean SD)

Left temporal

33.33 14.28
10 (33.33 %)

35.83 11.19
NA

MRI

Right temporal

11 (36.67 %)

NA

Bilateral temporal

9 (30.00 %)

NA

Normal
Hippocampal sclerosis

14 (46.67 %)
9 (30.00 %)

NA
NA

Encephalomal acia foci

Lacunar infarction

1 (3.33 %)
4 (13.33 %)

NA
NA

Encephalomal atrophy

2 (6.67 %)

NA

Disease course

More than 10 years

less than 10 years

10 (33.33 %)
20 (66.67 %)

NA
NA

Dug reaction

Drug-resistant
Drug-responsive

10 (33.33 %)
20 (66.67 %)

NA
NA

Normally, members from the same miRNA family play

similar physiological functions [27]. Family classifications
of miRNA were also downloaded from miRBase [25].
According to chromosome positional relationships
between miRNAs, any two miRNAs with maximum
distance less than defined distance were considered to be
in the same cluster. Members of a particular miRNA
cluster are highly likely to be processed as cotranscribed
units [28]. We derived human miRNA clusters from
miRBase [25]. 216 miRNA clusters which contain 644
miRNAs were found when maximum inter-miRNA distance
was set to 50 kb.
LncRNA Annotation Data and Classification
Human lncRNA annotation data were retrieved from
GENCODE Release 19 (GRCh37.p13) [29]. These lncRNAs
can be further reclassified into the following locus biotypes
based on their location with respect to protein-coding genes:
lincRNA, antisense, sense_intronic, sense_overlapping, and
processed_transcript [30] .
Reannotating the Infinium 450K Array to Construct
lncRNA and miRNA Methylation Profiles
We downloaded the human lncRNA annotation files and the
human primary miRNA annotation files as mentioned above.
Moreover, the lncRNA and miRNA promoters were defined
as 10 kb upstream from the transcriptional start site (TSS) of
each lncRNA or pri-miRNA [31, 32]. For each sample, we
constructed lncRNA or miRNA methylation profiles using the
methylation levels of the probes mapped into the promoter
region of the lncRNAs or miRNAs. The mapping procedure

was conducted using the bedtools. Since DNA methylation in

the proximal promoter of miRNAs is tightly linked to transcriptional silencing, as it is with protein-coding genes [33].
We used only the probes proximal to each TSS to determine
the DNA methylation status of lncRNA or miRNA promoters
[31].
MiRNA Target Genes and Functional Enrichment
Analysis
Human miRNA target data was obtained from an integrated
miRNA target predicting tool, namely miRGator v3.0
(http://mirgator.kobic.re.kr/) [34]. It contains validated target
databases that are available at miRecords, Tarbase, and
miRTarBase and many target prediction methods including
TargetScan, microRNA.org, miRBase, PITA, PicTar, and
miRDB. We picked out the target gene assemblages of each
miRNA by extracting only miRNA target pairs that were
predicted by at least four tools. Annotation on the target
gene candidates of differentially methylated miRNAs was
performed using DAVID Bioinformatics Resources 6.7
(http://david.abcc.ncifcrf.gov/) [35, 36] to describe the gene
ontology (GO) [37] and Kyoto encyclopedia of genes and
g e n o m e s ( K E G G ) [ 3 8 ] p a t h w a y. A d j u s t e d p
(Benjamini) < 0.05 was set as the threshold of significance
for GO terms and KEGG pathways.
Predicting the Functions of lncRNAs
We used lncRNA2function (http://mlg.hit.edu.cn/lncrna2
function/) to predict the functions of lncRNAs. The
lncRNA2function is an important resource for further
investigating the functions of a single human lncRNA, or

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functionally annotating a set of human lncRNAs of interest,

which based on the hypergeometric test to functionally
annotate a single lncRNA or a set of lncRNA genes with
significantly enriched functional terms among the proteincoding genes that are co-expressed with the lncRNAs. The
functional terms include all nodes in the GO and 4380 human
biological pathways collected from 12 pathway databases
[39]. The enriched pathways and GO categories were reported
as significant only if the p value after multiple testing corrections (Benjamini) was <0.05.
Predicting miRNAlncRNA Interactions
Predicted miRNAlncRNA interactions data were
downloaded from starBase (http://starbase.sysu.edu.cn/) [40]
, which analyze a large set of Ago and RBP binding sites
derived from all available CLIP-Seq experimental techniques.
Statistical Analysis of Differential Methylation
We used Students t test for contrasting TLE samples with
healthy controls to obtain unadjusted p value, and a false discovery rate (FDR) analysis was used to correct for multiple
testing. A FDR less than 5 % (q < 0.05) was considered significant. While Students t test for contrasting clinical subgroup was used to determine significant differential methylation loci with unadjusted p value cutoff as <0.01 for lncRNA
and miRNA, all calculations were performed with the software R. Hierarchical clustering (HC) analysis was performed
with the MultiExperiment Viewer (MeV).

Results
Reannotation for Constructing DNA Methylation Profiles
of lncRNAs and miRNAs
To characterize DNA methylation patterns for lncRNAs and
miRNAs, we employed a computational strategy to reannotate
data of Infinium 450K arrays into human lncRNA- and
miRNA-associated promoter regions (the regions 10 kb upstream from the TSSs). In total, there were 61,784 probe sequences that corresponded to 8533 lncRNA promoter regions,
while 14,019 probe sequences for 1722 miRNA promoter
regions. On average, each lncRNA or miRNA had seven to
eight probe sequences mapped to corresponding promoter region. Although a substantial set of probe sequences mapped to
the regions 10 kb upstream from the TSSs, we retained only
the probes closest to each TSS to determine the DNA methylation status of lncRNA and miRNA promoters (8533 and
1722, respectively) (Fig. 1).

Identification of Differentially Methylated miRNAs

and lncRNAs
To further investigate the possible biological implication of
differential methylation, we focused on the subset of only
the proximal probes of lncRNA and miRNA promoters. In
lncRNA promoters, significantly differentiated methylated
(DM) loci between TLE and healthy control were observed
in 2.3 % of the CpG sites analyzed (194/8533) (with
FDR < 0.05). Among these loci, 168 (87 %) were
hypermethylated, compared to 26 (13 %) loci that were
hypomethylated (Supplementary Table S1). In miRNA promoters, significant differentially methylated loci between TLE
and healthy control were observed in 4.8 % of the CpG sites
analyzed (82/1722) (with FDR < 0.05), with 70 (85 %)
hypermethylated and 12 (15 %) loci hypomethylated
(Supplementary Table S2). The differentially methylated
lncRNAs and miRNAs were used for unsupervised HC analysis. The actual methylation levels for each probe sequence
are shown by heat maps (Fig. 2).
Classification of Differentially Methylated miRNAs
and lncRNAs
The majority of differentially methylated lncRNAs in TLE
were from antisense to protein-coding loci (antisense, 45 %),
intergenic regions (LincRNA, 44 %), or within introns of
protein-coding genes (sense_intronic, 5 %), with the others
representing overlapping transcripts from exons or introns in
both sense and antisense strand (Fig. 3a and Supplementary
Table S1). The differentially methylated miRNAs in TLE included novel miRNAs (45 %) and known miRNAs (55 %)
(Fig. 3b and Supplementary Table S2).
Interestingly, we identified several miRNA families and
clusters being differentially methylated between TLE and
healthy control, including three miRNA families and three
chromosomal clusters hypermethylated in TLE: one chromosomal cluster, mir-23b and mir-27b, from chromosome 9; let7 family comprising of miRNAs from one chromosomal cluster, let-7a, let-7d, and let-7f from chromosome 9; miR-515
family comprising of miRNAs from one chromosomal cluster,
mir-517b, mir-518a, and mir-516a, from chromosome 19; and
miR-548 family comprising of mir-548j from chromosome 22
and mir-603 from chromosome 10 (Fig. 3c).
LncRNA Functional Enrichment Analysis Results
To explore the potential functions of the differentially methylated lncRNA genes, we annotate lncRNA genes with significantly enriched functional terms among the protein-coding
genes that are co-expressed with the lncRNAs. Among the
input set of 194 lncRNAs with significantly differential methylation in the TLE relative to healthy control, a sum of 5424

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Fig. 1 Computational strategy
for reannotating Infinium 450K
array data to construct lncRNA
and miRNA methylation profiles
in TLE

Fig. 2 Unsupervised hierarchical

clustering analysis of miRNAs (a)
and lncRNAs (b) differentially
methylated in TLE relative to
healthy control. Values from
samples are presented
horizontally, left for healthy
controls while right for TLE
patients. MiRNAs and lncRNAs
are listed vertically. Green and red
represent levels of
hypomethylation and
hypermethylation, respectively,
while black indicates methylation
was not detected

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Fig. 3 Classification of differentially methylated miRNAs and lncRNAs.

a Pie graphs illustrating the rate distribution of differentially methylated
lncRNAs relative to their location with respect to protein-coding genes. b
Pie graphs illustrating the rate distribution of differentially methylated

miRNAs relative to annotated miRNA gene type. c Classified

differentially methylated miRNAs into miRNA family or cluster. Blue
and yellow represent miRNA family and miRNA cluster, respectively

co-expressed protein-coding genes was identified to be associated with 170 lncRNAs (24 lncRNAs could not be found in
the Ensembl v70 (GENCODE v15) and was excluded from
the enrichment analysis). The results showed that GO terms
were enriched significantly (Benjamini adjusted p value
<0.05), which are related to ion channel complex, synapse
and neuron part (cellular component), and ion/gated channel
activity, GABA receptor activity (molecular function), and
synaptic transmission, transmission of nerve impulse, and
neurological system process (biological process).
Significantly enriched pathway includes neuroactive ligandreceptor interaction, neuronal system, voltage-gated potassium channels, drug metabolism of cytochrome P450, neurotransmitter release cycle, and GABAergic synapse (Fig. 4a
and Supplementary Table S3).

mitogen-activated protein kinase (MAPK) signaling pathway

and neurotrophin signaling pathway (Fig. 4b and
Supplementary Table S4).

MiRNA Functional Enrichment Analysis Results

To identify the biological processes or pathways potentially
regulated by differentially methylated miRNA genes, we applied miRNA target prediction algorithm and functional annot a t i o n a n a l y s i s , m i R G a t o r d a t a b a s e a n d D AV I D
Bioinformatics Resources, which got a number of the putative
miRNA targets and assigned to enriched gene ontology terms
and pathways in KEGG. Finally, 82 miRNAs and 2222
miRNA target genes were obtained. The results showed that
GO terms were enriched significantly, which are related to
neuron projection and synapse (cellular component); transcription regulator activity and protein kinase activity (molecular function); and neuron projection development, neuron
differentiation, neuron development, and axon guidance (biological process). Significantly enriched pathway includes

A Set of Differentially Methylated miRNAs and lncRNAs

Was Found in Different Clinical Subgroups
According to clinical information, TLE patients can be divided into different subgroups: drug-resistant or drug-responsive,
MRI negative or hippocampal sclerosis, and disease course
>10 or <10 years. Two-sample t test was used to investigate
if methylation differences implicate in lncRNAs and miRNAs
between different subgroups. We found a set of miRNAs and
lncRNAs with significantly differential methylation (P < 0.01)
in the different subgroups (Supplementary Table S5).
Significantly differential methylation miRNAs include
MIR30A, MIR219, MIR128, MIR203, and MIR302, which
are reportedly dysregulated in the previous epilepsy researches. Thereinto, MIR219 can be found differentially
methylated both in drug-resistant or drug-responsive, MRI
negative and hippocampal sclerosis subgroup. Moreover, we
searched aberrant methylation of lncRNA or miRNA in
starBase and obtained two predicted miRNA-lncRNA interactions: RP11-361F15.2/MIR203 and RP11-361F15.2/
MIR30A.

Discussion
The present study is the first genome-wide analysis of
lncRNA and miRNA methylation in the peripheral blood of
patients with TLE. Our pilot study reveals that a limited

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Fig. 4 Enriched KEGG pathway
and GO annotation of genes
targeted by differentially
methylated miRNAs (a) and
lncRNAs (b). Genes targeted by
differentially methylated
miRNAs were predicted and
annotated with DAVID for the
categories molecular function,
biological process, cellular
component, and KEGG pathway.
The lncRNA2function is used for
functionally annotating human
lncRNAs of differentially
methylated and the functional
terms include all nodes in the GO
and biological pathways

number of lncRNAs and miRNAs display altered methylation

profiles in TLE and different clinical subgroups. With 87 %
(168/194) lncRNAs and 85 % (70/82) miRNAs were
hypermethylated, hypermethylation of lncRNA and miRNA
promoters is the major difference between TLE patients and
healthy controls, which is consistent with findings in hippocampus of TLE patients and rodent models of TLE where
hypermethylation of gene promoters was also the predominant effect [23, 41].
NcRNAs and Coding Genes Coordinately Mediate
Pathway Dysregulation in TLE
Epileptogenesis is a complex dynamic biological process that
may involve multiple epigenetic alterations [42].
Understanding this complex network might be implicated by

methylation and ncRNAs in epileptogenesis and development. We found that aberrant methylation of either lncRNA
or miRNA can perturb specific common pathways, such as
MAPK signaling pathway, neurotrophin signaling pathway,
and drug metabolism of cytochrome P450, indicating that
ncRNAs mediate the dysregulation of these pathways in a
coordinated manner (Fig. 5).
As the most enriched pathway, MAPK pathway is a highly
conserved module that controls fundamental cellular processes,
such as growth, proliferation, differentiation, migration, and
apoptosis [43]. In the nervous system, p38 MAPK signaling
has diverse functions beyond the control of cell death and survival and may control neuronal function such as synaptic plasticity [44, 45]. Additionally, Shao et al. found that p38 MAPK
signaling pathway was involved in pharmacoresistance of refractory epilepsy through the regulation of P-glycoprotein

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Fig. 5 The schematic diagram of miRNAs and lncRNAs cooperatively

mediated pathways dysregulation in TLE. The yellow rectangles
represent the key genes and blue circles and green rectangles for
differentially methylated miRNAs and lncRNAs, respectively. The lines
between them show the dysregulation of miRNA and/or lncRNA to
genes. Predicted or validated miRNA-mediated regulations and
lncRNA-mediated regulations with literature reported were linked.
Dotted lines between miRNA and lncRNA represent predicted
miRNAlncRNA interactions. The peripheral large circles or
rectangles denote the enriched pathways

gene, encoding the apoptosis regulator protein, bcl-2, which

can promote cell survival [54]. In rat model of TLE, upregulated miR-34a may participate in promoting neuronal death or
apoptosis and reduce neuronal survival by targeting bcl-2
[55]. Another example is tumor protein p53 (TP53).
Prolonged seizures lead to accumulation of p53 in the neurons
and inhibition or deficiencies of p53 are protective in epilepsy
models of seizure-induced neuronal death [56, 57].
Methylation-sensitive MEG3 could upregulate Bcl-2 via its
competing endogenous RNA activity, which was important
for MEG3 to regulate gastric cancer progression [58].
Additionally, MEG3 can function as a novel lncRNA tumor
suppressor by inducing accumulation of p53 in cancer [22].
Taken together, these results suggest that MEG3 could regulate
cancer development by targeting apoptosis-associated genes
TP53 and BCL-2. What is more, TP53 and BCL-2 can be a
target of several aberrant methylation miRNAs, such as let-7
and miR-23b. It is speculated that these apoptosis-associated
genes and pathway target by aberrant methylation of lncRNA
and/or miRNA may hint an ongoing struggle between cell
death and survival mechanisms in epileptogenesis and development in TLE (Fig. 7).
Epigenetic Pathomechanisms Underlying Drug Resistance
in TLE

expression [46]. In our study, most genes as revealed were

targeted by aberrantly methylated miRNAs or lncRNAs in this
pathway (Supplementary Fig. S2). Some of these genes, such
as calcium channel (CACN) genes and myocyte enhancerbinding factor 2C (MEF2C), were implicated in epilepsy or
seizure phenotype. Epilepsy patients and animal models of epilepsy studies have identified gain-of-function mutations in several CACN genes, stating the role of calcium channels in epilepsy pathophysiology [47]. CACN genes can be target of several aberrant methylation miRNAs, such as let-7a, miR-27b,
miR-23b, and miR-190b. For instance, CACNA1A is a member of the voltage-dependent calcium channel family. A novel
point mutation of which results in the impairment of the brain
calcium channel CaV2.1 and may have a central role in the
pathogenesis of epilepsy [48]. MEF2C, possible target of aberrant methylation miR-23b, is essential for early neurogenesis,
neuronal differentiation, and migration. Point mutations or deletions of MEF2C are responsible for severe mental retardation
and seizures [49, 50]. Another dysregulated pathway is
neurotrophin signaling pathway (Fig. 6), which transmits both
positive signals like enhanced survival and growth and negative
signals like neuronal apoptosis. These signals play an important
role for neural development and additional higher-order activities such as learning and memory [51, 52]. Neurotrophin signaling pathway was also reported by Wang et al. in their transcriptome analysis of circulating miRNA in epilepsy patients
[53]. We found that the lncRNA and miRNA regulated key
components of this pathway. One such example is the BCL2

TLE is the most common epilepsy syndrome in drug-resistant

epilepsy [59]. There are several proposed mechanisms for the
acquisition of drug resistance in our study. Valproic acid
(VPA) and gabapentin are frequently used antiepileptic drugs
whose antiepileptic properties have been partly attributed to
the inhibition of ion channels including voltage-dependent
CACN [60]. Aberrant methylation miRNA (miR-27b, let-7a,
etc.) can target voltage-dependent CACN, which may alter
antiepileptic properties of calcium channel blocker.
Significantly enriched pathway of aberrant methylation
lncRNA includes drug metabolism of cytochrome P450,
which plays a critical role in the metabolism of 12 drugs including some the antiepileptics: felbamate, carbamazepine,
oxcarbazepine, and VPA. What is more, miR-27b level may
affect cytochrome P450 3A activity [61]. From the above,
changes in the enzyme systems responsible for drug metabolism and drug targets could be regulatory factors. There is also
evidence that endogenous miRNAs can modulate the function
of bloodbrain barrier (BBB) [62, 63]. With aberrant methylation of miR-27b and miR-30a, which were found to increase
BBB function in patients with multiple sclerosis [64], BBB
dysfunction could also be found to be during TLE and
pharmacoresistance [65, 66].
Undeniably, there are several limitations of the present
study that should be discussed. First, due to the small sample
size, aberrantly methylated miRNAs and lncRNAs should be
replicated in a larger cohort, maybe collecting samples from

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Fig. 6 A depiction of neurotrophin signaling pathway dysregulation in

KEGG database. miRNAs and lncRNAs cooperatively mediated
neurotrophin signaling pathway dysregulation in TLE. The
representative component of pathway is shown. Proteins or complexes
targeted by differentially methylated miRNAs and/or lncRNAs are

Fig. 7 A model illustrating the

misregulated processes
intertwined by DNA methylation,
ncRNAs, and coding genes.
lncRNAs and miRNAs
cooperatively mediate the
pathway dysregulation which
may be implicated in
epileptogenesis and
pharmacoresistance mechanisms
in TLE

indicated in red character. The lines between them show the

dysregulation of miRNA and/or lncRNA to genes. Predicted or
validated miRNA-mediated regulations and lncRNA-mediated
regulations with literature reported were linked

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diverse neurological research centers. Second, we had no technical verification for Human Methylation 450K BeadChip
data, but studies on the validity of the Infinium 450K have
shown good congruence with pyrosequencing data [67].
Moreover, in our previous study, we had selected a subset of
differentially methylated CpG loci for additional methylation
analysis using the pyrosequencing method, which was well
correlated with Methylation 450K BeadChip test (data unpublished). Third, although we did not collect samples for quantification of RNA (lncRNA and miRNA) in whole blood,
previously published studies showed lncRNA and/or
miRNA expression changes were associated with promoter
methylation events [21, 23, 31]. Finally, although in this study
we have found aberrantly methylated miRNAs and lncRNAs,
it remained unsolved whether the changes in miRNA and
lncRNA methylation represent a cause or a consequence of
the epileptogenic process. Further studies in animal models of
TLE and brain tissues of TLE patients may help to resolve
these issues.

6.

7.

8.

9.
10.

11.

12.
13.

14.
15.

Conclusion
In summary, we studied the functions and mechanisms of
DNA methylation of ncRNAs in human TLE. The identified
TLE-associated or clinically relevant ncRNAs could be further evaluated for use as TLE and drug resistance biomarkers
and potential therapeutic targets.
Acknowledgements This study was supported by the National Natural
Science Foundation of China (NNSFC), grant no. 81401078 to H.Y.L and
no. 81371435 to B.X.

16.

17.

18.

19.
20.

Compliance with Ethical Standard

Conflict of Interest The authors declare that they have no conflict of
interest.

21.

22.
23.

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