miRNA expression analysis in cortical dysplasia: Regulation of mTOR and LIS1 pathway

Epilepsy Research (2014) 108, 433—441

journal homepage: www.elsevier.com/locate/epilepsyres

miRNA expression analysis in cortical dysplasia: Regulation of mTOR and LIS1 pathway Ji Yeoun Lee a,1, Ae-Kyung Park b,1, Eun-Sun Lee a, Woong-Yang Park c,d, Sung-Hye Park e, Jung Won Choi a, Ji Hoon Phi a, Kyu-Chang Wang a, Seung-Ki Kim a,∗ a

Division of Pediatric Neurosurgery, Seoul National University Children’s Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea b College of Pharmacy, Sunchon National University, Jeonnam, Republic of Korea c Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea d Translational Genomics Laboratory, Samsung Genome Institute, Samsung Medical Center, Seoul, Republic of Korea e Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea Received 24 June 2013; received in revised form 11 December 2013; accepted 14 January 2014 Available online 30 January 2014

KEYWORDS Cortical dysplasia; miRNA; Microarray; mTOR; LIS1

Summary Cortical dysplasia (CD) is a common cause of epilepsy in children and is characterized by focal regions of malformed cerebral cortex. The pathogenesis and epileptogenesis of CD have not been fully elucidated, and in particular, the potential role of epigenetics has not been examined. miRNA microarray was performed on surgical specimens from CD (n = 8) and normal control (n = 2) children. A total of 10 differentially expressed miRNAs (DEmiRs) that were up-regulated in CD were identified including hsa-miR-21 and hsa-miR-155. The microarray results were validated using quantitative real-time PCR. After searching for the putative target genes of the DEmiRs, their biological significance was further evaluated by exploring the pathways in which the genes were enriched. The mammalian target of rapamycin (mTOR) signaling pathway was the most significantly associated, and the pathway of lissencephaly gene in neuronal migration and development was also noted.

∗

Corresponding author at: Division of Pediatric Neurosurgery, Seoul National University Children’s Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea. Tel.: +82 2 2072 3084; fax: +82 2 744 8459. E-mail address: [email protected] (S.-K. Kim). 1 These authors contributed equally to the manuscript. 0920-1211/$ — see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2014.01.005

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J.Y. Lee and A.-K. Park et al. This study suggests a possible role for miRNAs in the pathogenesis of CD, especially in relation to the mTOR signaling pathway. Future studies on the epigenetic mechanisms underlying CD pathogenesis and epileptogenesis are needed. © 2014 Elsevier B.V. All rights reserved.

Introduction Cortical dysplasia (CD) is a subgroup of malformations in cortical development (MCD) characterized by anatomic disorganization of the cellular layers and the presence of morphologically abnormal cells (Bentivoglio et al., 2003; Blumcke et al., 2009). With the advancement of magnetic resonance imaging (MRI) of the brain, the ease of diagnosis of MCD has increased greatly, and CD is the most frequently detected MCD (Bronen et al., 1997; Cotter et al., 1999; Gomez-Anson et al., 2000; Palmini et al., 1994). Hence, epilepsies previously thought to be cryptogenic are now recognized to be secondary to CD, which is one of the most common causes of medically intractable epilepsy in children (Krsek et al., 2008; Palmini et al., 1994). Surgical resection is the treatment of choice for these lesions, but the outcome is not always satisfactory. There is a great clinical need for research on CD pathogenesis and the mechanisms underlying its epileptogenicity. In search for the pathogenesis of CD, studies on its genetic etiology have been performed with a focus on a neurodevelopmental origin. Additionally, the discovery of the causal gene in other subtypes of MCD such as tuberous sclerosis (TSC) or the link between hemimegalencephaly (HME) and genetic syndromes have supported the possibility of a molecular genetic etiology of CD (Crino, 2009). However, most of these studies have focused on genetic mutations (Becker et al., 2002; Fassunke et al., 2004) or aberrant gene expression (Kim et al., 2003), and no studies examining epigenetics are available. This study aimed to evaluate the miRNA expression in CD and to compare that to normal brain cortex miRNA expression to look for a post-translational explanation of CD pathogenesis and epileptogenicity.

Methods Patients Surgical specimens were obtained during epilepsy surgery from patients with medically intractable epilepsy. The patients (n = 15; mean age at surgery: 8 years) were diagnosed with CD (type I: 8, type II: 7; Table 1). The normal cortex was obtained during surgery for deep seated lesions (Table 2). The diagnoses were confirmed by a pathologist (PSH) according to the recent CD classification scheme (Blumcke et al., 2011). Four cases (mean age at surgery: 6 years) with a histologically normal cortex were included as normal controls. For the miRNA microarray, 8 CD samples (mean age at surgery: 9 years) and 2 normal controls (ages: 7 and 13 years) were used. Then, to validate the microarray results, the expression levels of the DEmiRs were confirmed by quantitative real-time PCR (qRT-PCR) using a different set of 8 CD samples (mean age at surgery: 7 years) and 3 normal controls (mean age at surgery: 6 years). CD case #13

and normal control #2 were used for both the microarray and qRT-PCR (Tables 1 and 2). All the samples were snap-frozen immediately after the resection and were stored at −80 ◦ C until use. Informed consent was obtained from the patients’ parents or guardians for the study, which was approved by the Institutional Review Board of the Seoul National University Hospital.

miRNA microarray chip processing and analysis of the miRNA expression data Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) with a small RNA enrichment procedure. An Agilent Human miRNA Microarray Kit (V3) was used as the miRNA microarray chip for the hybridization. The RNA labeling and hybridization were performed according to the manufacturer’s instructions. The microarray images were scanned with an Agilent microarray scanner. The total gene signals were extracted using the Agilent Feature Extraction software and were further log2 -transformed. To identify the differentially expressed miRNAs (DEmiRs) between the normal brain tissue and the CD, Bayesian moderated t-statistics were computed (Smyth, 2004), and the analysis was confined to the miRNAs whose expression signals were defined as ‘‘detected’’ by the Agilent Feature Extraction software in all the samples. The miRNAs were defined as significantly upor down-regulated if their t-test p values were less than 0.05 and their fold changes were greater than 2-fold. An unsupervised hierarchical clustering analysis was performed with the standardized expression values using the ‘‘Manhattan’’ distance metric and the ‘‘Ward’’ linkage algorithm.

Validation of the DEmiR by quantitative Real-Time PCR The DEmiRs identified by the microarray were validated using TaqMan probes (Applied Biosystems, Carlsbad, CA) using the Applied Biosystems 7500 Real-time PCR system. The cDNAs were prepared with the High-Capacity cDNA Synthesis Kit (Applied Biosystems) using 10 ng of the total RNA as a template. The miR sequence-specific reverse transcription-PCR (RT-PCR) primers for hsa-miR-21, 130b, 155, 193b, 199b and the endogenous control RNU6B were used (Ambion). The reactions were performed in triplicate according to the TaqMan Gene Quantitation assay protocol. Fischer’s exact test was performed for statistical evaluation. P-value less than 0.05 was considered significant.

Pathway analysis with experimentally verified target genes Using Tarbase 6.0, we identified the experimentally confirmed human target genes for the up- and down-regulated miRNAs. To exclude the genes expressed at low levels in

miRNA in cortical dysplasia Table 1

435

Demographics and clinical features of patients.

Case number

Age (year)/sex

Seizure type

Location

Type of surgery

Pathologya

Use

1 2

16/M 5/M

CPS CPS

Lt occipital Lt temporal

Type Ia Type Ib

Microarray Microarray

3 4

16/F 15/M

CPS with SG GTC

Lt frontal Lt temporal

Type Ia Type Ia

Microarray qRT-PCR

5

11/M

CPS with SG

Lt temporal

Type Ia

qRT-PCR

6 7 8 9 10 11 12 13

11/F 8/M 2/F 4/F 2/F 3/F 15/F 8/M

CPS CPS CPS GTC GTC CPS CPS CPS

Lt occipital Rt frontal Rt temporal Lt parietal Rt temporal Rt frontal Lt parietal Rt frontal

Lesionectomy Standard temporal lobectomy Lesionectomy Standard temporal lobectomy Standard temporal lobectomy Lesionectomy Lesionectomy Lesionectomy Lesionectomy Lesionectomy Lesionectomy Lesionectomy Lesionectomy

Type Type Type Type Type Type Type Type

14

1/M

CPS

Rt hemisphere

Type IIb

15

1/M

GTC

Rt frontal

Functional hemispherectomy Lesionectomy

Microarray qRT-PCR qRT-PCR qRT-PCR Microarray qRT-PCR Microarray Microarray, qRT-PCR Microarray

Type IIb

qRT-PCR

Ib Ib Ib IIa IIa IIa IIb IIb

M: male; F: female; CPS: complex partial seizure; SG: secondary generalization; GTC: generalized tonic clonic seizure; Lt: left; Rt: right; qRT-PCR: quantitative Real-Time PCR. a Pathologic diagnosis according to ILAE classification (Blumcke et al., 2011).

normal human brain among the target genes, we used publicly available microarray data; mRNA expression data from normal human brains from the dissected post-mortem superior frontal gyrus were acquired from the National Center for Biotechnical Information Gene Expression Omnibus ftp site (accession numbers: GSE17757). This dataset included 23 human brain samples with a wide range of ages, but we used only the expression data from 11 samples of a young age (≤17 years old) to match the ages with our samples. The raw data were pre-processed using a Robust Multi-Array Average (RMA) approach and combined with the miRNA target gene list. Then, we retrieved the final target genes of the DEmiRs by excluding the genes with low expression levels in the normal human brains (average of log2 expression of 11 samples < 7). To identify the signaling pathways deregulated by the up- or down-regulated miRNAs in CD, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) and

Table 2

the BioCarta pathways in the DAVID bioinformatics resources (http://david.abcc.ncifcrf.gov/). All the statistical analyses were performed using R (http://www.R-project.org) and Bioconductor software (http://bioconductor.org).

Results Differential miRNA expression between cortical dysplasia and normal brain tissue To identify the miRNAs that are differentially expressed in CD, we first performed pairwise comparisons between 3 groups, normal brain, CD type I, and CD type II. In comparisons with the normal brain tissues, more significantly different miRNAs were detected in CD type II than in type I (Fig. 1A and B). However, it was revealed that the expression

Demographics and clinical features of control.

Case number

Age (year)/sex

Location

Pathology of main lesion

Use

1 2 3 4

7/M 13/M 3/M 2/F

Rt medial temporal Rt frontal Lt medial temporal Lt medial temporal

Choroidal fissure cyst Extraventricular neurocytoma Ganglioglioma Ganglioglioma

Microarray Microarray qRT-PCR qRT-PCR qRT-PCR

M: male; F: female; Rt: right; Lt: left; qRT-PCR: quantitative real-time PCR.

436

J.Y. Lee and A.-K. Park et al.

Figure 1 Heatmap and unsupervised hierarchical clustering of the differentially expressed miRNAs in cortical dysplasia (CD). (A) Differentially expressed miRNAs between normal and type I CD. (B) Differentially expressed miRNAs between normal and type II CD. (C) Differentially expressed miRNAs between normal and CD.

Table 3

Differentially expressed miRNAs in cortical dysplasia compared to normal human brains.

miRNA

Up-regulated in CD hsa-miR-193b hsa-miR-155 hsa-miR-193a-3p hsa-miR-199b-5p hsa-miR-532-5p hsa-miR-130b hsa-miR-21 Down-regulated in CD hsa-miR-139-3p hsa-miR-877 hsa-miR-572

Average log2 expression value of 10 samples

Type I & II vs normal

Type II vs normal

Type I vs normal

log2 FC

log2 FC

log2 FC

p value

p value

p value

6.3 4.7 5.8 5.2 4.7 6.5 12.7

1.4 1.9 2.2 2.4 1.4 1.0 2.1

0.004 0.011 0.016 0.033 0.037 0.041 0.049

1.6 2.1 2.6 2.8 1.7 1.1 1.6

0.004 0.014 0.003 0.028 0.013 0.037 0.059

1.2 1.7 1.9 2.0 1.0 0.8 2.6

0.033 0.009 0.050 0.009 0.134 0.143 0.032

7.3 6.2 7.9

−1.0 −1.2 −1.1

0.021 0.038 0.041

−1.0 −0.9 −0.8

0.073 0.129 0.117

−1.1 −1.6 −1.3

0.039 0.042 0.037

CD: cortical dysplasia; FC: fold-change.

miRNA in cortical dysplasia

437

Putative deregulated signaling pathways targeted by differentially expressed miRNAs in cortical dysplasia

Figure 2 qRT-PCR confirmation of the DEmiR expression in cortical dysplasia (CD) and normal controls. The miRs are highly expressed in CD, compared with normal for has-miR21, hsa-miR-130, hsa-miR-155, hsa-miR-193b, and hsa-miR-199. *p < 0.05.

of only 1 miRNA (hsa-miR-877) was significantly different between type I and type II (p value: 0.044); therefore, we decided to focus on the DEmiRs between the normal brain tissue and CD that were detected in the analysis without regard to CD type. We identified 7 miRNAs that were more than 2-fold up-regulated in CD and 3 miRNAs that were more than 2-fold down-regulated in CD compared with the normal human brain tissues (Table 3 and Fig. 1C). We found that hsa-mir-21 is highly expressed in all 10 samples including cortical dysplasia and normal brain (average log2 expression value: 12.7), while the expression of the other DEmiRs was considerably lower than that of the hsamir-21; the mean expression levels were several tens- to hundreds-fold lower in our samples (Table 3). Two miRNAs out of the 7 up-regulated miRNAs, hsa-mir-21 and hsa-mir155, are well known oncomiRs that are over-expressed in various cancers and related to cancer formation, invasion, and metastasis (Asangani et al., 2008; Meng et al., 2007; Papagiannakopoulos et al., 2008). A large number of genes including many tumor suppressors have been validated as target genes for these 2 miRNAs by various experimental procedures. In addition, it has been shown that increased levels of hsa-miR-21 are also related to the development of heart disease (Thum et al., 2008), while hsa-miR-155 is involved in the inflammatory response and in innate immunity (Teng and Papavasiliou, 2009). Except for these 2 miRNAs, relatively little is known about the other 5 up-regulated and 3 down-regulated miRNAs. The microarray results were validated using qRT-PCR. The DemiRs were confirmed to show different expression between the normal control and CD samples, in correlation with the microarray results (Fig. 2).

To detect the putative signaling pathways that are deregulated by the DEmiRs in CD, we collected the experimentally confirmed human target genes of the DEmiRs from the TarBase database (version 6.0). Out of 3 down-regulated miRNAs, no experimentally proven target for has-miR-877 and has-miR-139-3p was detected, and only 1 gene (CDKN1A) was identified as a target for hsa-miR-572 alone; therefore, further analysis was confined to the target genes for the up-regulated miRNAs. In addition, from the retrieved target genes, we excluded the genes that are expressed in normal human brain tissues at low levels based on the publicly available mRNA expression data from the normal human brain tissues. Finally, we obtained a total of 924 target genes for 6 out of the 7 up-regulated miRNAs, and these target genes were analyzed using the DAVID informatics resources to identify the signaling pathways that are considered to be deregulated by the up-regulated miRNAs in CD. A total of 12 signaling pathways were recognized to be significantly enriched in the target genes of the up-regulated miRNAs in CD (Table 4). The most enriched pathway was the mammalian target of rapamycin (mTOR) signaling pathway, which remained significant even after Bonferroni corrections for multiple comparisons (Fig. 3). We also found that the lissencephaly gene in neuronal migration and development (LIS1 pathway, Fig. 4) was one of the pathways deregulated by the up-regulated miRNAs. In addition, the WNT and Notch signaling and Adherens junction pathways were noted to be significantly altered in CD (Table 4).

Discussion This study revealed the potential epigenetic mechanism underlying the pathogenesis and epileptogenesis of CD through miRNA microarray. Seven up-regulated DEmiRs were found, among which hsa-miR-21, and hsa-miR-155 were noted for their association with cell proliferation, cell differentiation, and inflammation. When the putative target mRNAs were annotated and analyzed by cell signaling pathway, the genes in the mTOR and LIS1 pathways were highly enriched. These pathways may provide an explanation for the 2 major features of CD, the mTOR signaling pathway for the dysmorphism of the cells and the LIS1 pathway for the dyslamination.

mTOR pathway The mTOR signaling pathway is known to mediate cellular growth, proliferation, metabolism, and survival through multiple downstream pathways (Fingar et al., 2002; Wong, 2010). The critical role of the mTOR pathway in the pathogenesis of MCD was revealed after the discovery of TSC1 and TSC2 as the causative genes for TSC (European Chromosome 16 Tuberous Sclerosis, 1993; van Slegtenhorst et al., 1997). Mutations in either TSC1 or TSC2 lead to the activation of mTOR, resulting in cell proliferation as well as abnormalities in neuronal migration, explaining the cytomegalic and

438 Table 4

J.Y. Lee and A.-K. Park et al. Pathways enriched in target genes of up-regulated miRNAs in cortical dysplasia.

Category

Term

Count

P value

Benjamini p value

Genes

KEGG

mTOR signaling pathway

12

0.00016

0.026

EIF4B, RSK, HIF1␣, PTEN, TSC1, LKB1, VEGF, RHEB, MO25, RICTOR, PIK3R1,

KEGG

RNA degradation

11

0.0015

0.11

PAPOLA, DCP1A, PNPT1, TTC37, EXOSC2, RQCD1, LSM3, SKIV2L2, CNOT6, CNOT4, DDX6

KEGG

Aminoacyl-tRNA biosynthesis

9

0.0022

0.11

YARS, CARS, NARS, RARS, LARS, HARS, EPRS, CARS2, MARS

KEGG

Pancreatic cancer

12

0.0028

0.11

KRAS, VEGFA, SMAD4, SMAD3, MAPK8, SMAD2, RB1, CDK4, STAT3, PIK3R1, TGFB2, AKT2

KEGG

Colorectal cancer

13

0.0033

0.10

MSH2, SMAD4, SMAD3, SMAD2, APPL1, TGFB2, CTNNB1, KRAS, GSK3B, MAPK8, PIK3R1, APC, AKT2

BIOCARTA

Presenilin action in Notch and Wnt signaling

6

0.0045

0.58

PSEN1, GSK3B, BTRC, RBPJ, CTNNB1, APC

KEGG

Biosynthesis of unsaturated fatty acids

6

0.0077

0.19

ACOT7, PTPLB, FADS1, SCD, HSD17B12, TECR

KEGG

Valine, leucine and isoleucine degradation

8

0.013

0.26

BCAT1, MUT, OXCT1, ACAT1, PCCB, ALDH3A2, ALDH9A1, HADHB

KEGG

Endometrial cancer

8

0.031

0.46

KRAS, GSK3B, CTNNA1, PTEN, PIK3R1, CTNNB1, APC, AKT2

KEGG

Adherens junction

10

0.035

0.50

PTPRJ, TJP1, SMAD4, SMAD3, CTNND1, ACTN1, SMAD2, SSX2IP, CTNNA1, CTNNB1

KEGG

Vitamin B6 metabolism

3

0.044

0.51

PDXK, PDXP, PSAT1

BIOCARTA

Lissencephaly gene (LIS1) in neuronal migration and development

4

0.047

0.99

NUDEL, CLIP170, ApoER, CDK5

KEGG: Kyoto Encyclopedia of Genes and Genomes.

dysplastic cells with dyslamination observed in the tubers in TSC (Wong, 2013). Because the pathologic features of CD are similar to the tubers in TSC, the role of the mTOR pathway in the pathogenesis and epileptogenesis of CD has been an investigated in depth (Wong, 2013). Although similar mutations in TSC1 or TSC2 were not as commonly found, the evidence for mTOR pathway activation in CD has been reported in gene expression studies (Kim et al., 2003) as well

as having been validated at the protein level (Baybis et al., 2004; Ljungberg et al., 2006; Miyata et al., 2004; Schick et al., 2007). Recently, whole-exome sequencing in HME has revealed somatic mutations in genes related to the mTOR pathway (Lee et al., 2012). Overall, the mTOR signaling pathway is currently the key player in the pathogenesis of the entire MCD disease spectrum. The importance of the mTOR pathway has been suggested further as the ‘‘primary’’

miRNA in cortical dysplasia

439

Figure 3 The mTOR signaling pathway is deregulated by the up-regulated miRNAs in cortical dysplasia. The putative target genes of the differentially expressed miRNAs are in green and the corresponding miRNA is written in red. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

signaling pathway triggering the various cellular and molecular mechanisms leading to epileptogenesis in diverse types of epilepsy and not only restricted to the diseases related to MCD (Wong, 2010). In this study, numerous upstream genes (RSK, PTEN, TSC1, LKB1, RHEB, MO25, RICTOR, PIK3R1, and AKT) and downstream genes (EIF4B, HIF1˛, VEGF) of the mTOR signaling pathway were found to be putative target genes of the DEmiRs. Several alternative ways for activation of the mTOR pathway other than the mutation in TSC1 or TSC2 may be proposed. First, TSC1 is marked as the target gene of a DEmiR up-regulated in CD; therefore, TSC1 may be underexpressed, leading to a decreased inhibitory effect on mTOR. Second, PTEN, which is an upstream gene in the mTOR pathway, may also be targeted by hsa-miR-21. It is

noteworthy that the animal model of PTEN deficiency is known to mimic the neurologic features of TSC mouse models including seizures, megalencephaly, and neuronal hypertrophy (D’Arcangelo, 2009; Kwon et al., 2003). Such epigenetic regulation of the mTOR pathway by a miRNA may provide an explanation for how the mTOR pathway may be hyperactivated through mechanisms other than a TSC mutation.

LIS1 pathway LIS1 is one of the causative genes of classic lissencephaly, and the LIS1 protein is a part of the dynein complex that is related to the microtubule motor protein (Dobyns et al.,

Figure 4 The LIS1 pathway is deregulated by the up-regulated miRNAs in cortical dysplasia. The putative target genes of the differentially expressed miRNAs are in green and the corresponding miRNA is written in red. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

440 1993). It is thereby associated in microtubule-based transport, making it crucial for neuronal migration (Liu et al., 2012). Several genes (NUDEL, CLIP170, ApoER1, and CDK5) related to LIS1 in neuronal migration and development were noted to be potential targets of the DEmiRs. A disturbance in LIS1 in the neuronal migration and development pathway may explain the dyslamination observed in CD. Indeed, starting from the pathological similarities between lissencephaly and CD, a possible role for microtubule-based transport has been implicated in the pathogenesis of CD (Liu et al., 2012; Wynshaw-Boris et al., 2010). The present study is limited in that the heterogeneous cellular population in CD was not considered. The heterogeneity of the abnormal cell population is a critical limitation in CD research (Palmini et al., 2004). Additionally, the different location of the lesions in the brains of the individual patients may have added to the inter-sample heterogeneity. This cellular heterogeneity is also an important obstacle in the field of microarray-based research (Cleator et al., 2006; Debey et al., 2004). Therefore, the intraand inter-sample variability should be taken into serious consideration in future studies involving microarray techniques with CD. In this regard, it should be noted that these variations may have accounted for the rather high heterogeneity of the miRNA expression between the CD samples in the present study. Separation through microdissection and the evaluation of balloon cells, cytomegalic neurons, etc. may be helpful. Additionally, the fundamental question of whether the changes in gene expression represent the causes or consequences of pathogenesis or epileptogenesis must be elucidated. Based on the results of the present study, functional validation should be performed in the future by confirming the DEmiRs in the tissues and by proving reciprocal expression of the putative targets in relation to the DEmiR. Ultimately, evaluation of the differences in the phenotype after a targeted change in expression of a DEmiR or its putative target genes would be necessary.

Conflict of interest The authors have no conflict of interest to disclose.

Acknowledgement This study was supported by a grant of the Korea Healthcare Technology R & D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A100620) and the Seoul National University Hospital Research Fund (30-2010-0120).

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