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DOI 10.1007/s12035-016-0353-x
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
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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Table 1 Clinical characteristics
of individuals
TLE
No.
Control
30
30
Male (n = 18)
25.28 11.75
28.17 13.96
Female (n = 12)
Inter-ictal EEG
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
1 (3.33 %)
4 (13.33 %)
NA
NA
Encephalomal atrophy
2 (6.67 %)
NA
Disease course
10 (33.33 %)
20 (66.67 %)
NA
NA
Dug reaction
Drug-resistant
Drug-responsive
10 (33.33 %)
20 (66.67 %)
NA
NA
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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).
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Fig. 1 Computational strategy
for reannotating Infinium 450K
array data to construct lncRNA
and miRNA methylation profiles
in TLE
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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).
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
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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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.
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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.
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