MicroRNAs as biomarkers in molecular diagnosis of refractory epilepsy

Epilepsy is a chronic neurologic disorder characterized by recurring abnormal electricity activity in brain, which is estimated to afflict about 65 million individuals world-wide [1]. Approximately 10 % of the publics have at least one seizure during their lifetime and among them about 30 % will develop epilepsy, suffering substantial deleterious influence on their health due to long-term use of drugs [2]. Nowadays it lacks a complete understanding of the epileptogenic process but it is thought to involve neuronal death or dysfunction, neurogenesis and others [2]. Many researchs now support miRNAs as a critical layer of gene expression regulation in epilepsy, which provides new putative targets for the control of seizures, seizure- induced damage and as potential biomarkers of epileptogenesis. miRNA is detected in many biofluids, including serum and CSF and it appears to be very stable [3, 4].

Current strategies to fight epilepsy focus on arresting epileptogenesis induced by brain trauma and other diseases through treatment of anti-epileptic drugs [3]. Substantial research has focused on the involvement of microRNAs in epilepsy, including screening the aberrant expression of microRNAs and searching their target genes. The isolation of miRNAs’ target genes would enable the development of a better understanding of the etiology, and the establishment of diagnostic biomarkers, and the identification of novel miRNA-based therapeutics.

Globally, the estimated proportion of the general population with active epilepsy is about 0.5-1 % and 20–30 % of those patients will develop a drug-resistant epilepsy (DRE) [4, 5]. Clinical trials demonstrated that cerebrovascular diseases, encephalitis and cortical dysplasia function as main causes of DRE. Diseases including West syndrome, Lennox-Gastaut syndrome and tuberous sclerosis could assist in identifying DRE [6]. Moreover, temporal lobe epilepsy (TLE) represents the most common syndrome in adults and often develops pharmacoresistence [7]. TLE frequently develops into DRE as a result of hippocampal sclerosis (HS) [8–10], inflammation [11], neurocysticercosis [12], brain insult [13], or malformations of cortical development [14, 15]. However, a consensus definition and prognosis for DRE remained elusive [16, 17]. In 2010, the International League against epilepsy (ILAE) addressed the need for a definition, declaring that“Drug resistant epilepsy may be defined as failure of adequate trials of two tolerated and appropriately chosen and used AED schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom” [18]. This proposal provided a precise definition of DRE but, left forming an efficacious prognosis method for DRE unexplored to this day. Past early-identification of DRE is mainly based on clinical manifestations like the number of seizures [19]. Other than applying the well-established electroencephalography (EEG) monitoring, other imaging testsing and clinical symptoms and signs watching, DRE urgently requires a set of molecular biomarkers to clarify the early prognosis, wherein microRNAs play an important role [20–22].

miRNA is a non-coding single-stranded RNA molecule with length of about 20–24 nucleotides and has highly conservatism, tissue specificity and temporality. It does not have open reading frame and mainly regulates expression of gene after transcription [23]. Most miRNA genes are present in the genome as the form of single copy, multiple copies or a gene cluster. Each miRNA can regulate one or more target genes, while several miRNAs can regulate the same target gene. miRNA regulates the expression level of proteins in cells through this complicated network [24]. Essentially, miRNA is a highly conserved non-coding single-stranded endogenous RNA molecule that plays an important role in the negative regulation of the target gene expression after transcription [25].

In animal experiments about DRE, researchers mainly focused on temporal lobe epilepsy and hippocampal sclerosis by artificially modeling mice or rats. Researchers further compartmentalized subgroups by distinguishing different time phases of seizures, different locations and different injury statuses of the brain.

Two studies published in 2012 on miRNA profiles in TLE revealed a considerable amount of dysregulated miRNAs in human diseased tissue. Kan et al. [36] focused on medial temporal lobe epilepsy (mTLE), a subtype of TLE caused by hippocampal sclerosis [37]. They performed genome-wide miRNA profiling in 20 human hippocampal tissue samples, including both sclerotic and non-sclerotic. The study also identified 51 strongly regulated miRNAs, wherein 11 miRNAs play essential roles. Of the 11 miRNAs, 7 were up-regulated- miR-190, miR-20a, miR-32, miR-374a, miR-26b, miR-193a-3p, miR-92b and 4 were down-regulated - miR-184, miR-138, miR-221, miR-222. Kan et al. found that miR-92b, miR-637, and miR-665 showed inconsistent localization between being prominent in the cytoplasm of neurons in autopsy controls and being in the nucleus of both neurons and astrocytes in mTLE samples. However, miR-138 expression was limited to the cytoplasm in mTLE patients. These findings together suggest that the localization of a specific miRNA may have significant clinical applications [38]. Consequently, detecting the nuclear expression of miR-92a, miR-637 and miR-665 may be a useful method for epilepsy diagnosis. Furthermore, 16 miRNAs involved in epileptogenesis were identified by summarizing the meaningful results from many studies, including miRNAs modulating neuronal maturation (miR-137 [39]), neural cell proliferation in cortex (miR-9 [40], miR-124 [41, 42], miR-137 [40, 43], miR-184 [40, 44, 45] and let-7b [40, 43, 46]), and neural differentiation (miR-153 [47], miR-324 [47], miR-181a [47], miR-34a [27, 48]). Other miRNAs are reported to be critical in neuronal migration (miR-9 [49], miR-134 [23], miR-137 [50]), neuronal differentiation (miR-125b [51] miR-137 [40]) and neural inflammation (miR-146a [52]).

Research on epilepsy-related microRNAs in human experiments typically progress slowly because factors key factors such as drug treatment influence miRNA profiling results. No clear result has been established on the relationship between microRNA and intractable epilepsy in human experiments, leaving a huge development space for applying microRNAs to potential biomarkers and therapeutic targets in refractory epilepsy. In this regard, it is a promising research direction to profile miRNA expression in both diseased brain tissue and corresponding plasm samples, and then compare fold changes of miRNA expressions to those in control groups.

The mechanism of down-regulation of a series of miRNA in TLE patients is well understood. McKiernan et al. [45] profiled miRNA expression in sclerotic hippocampi from TLE patients and found a significant down-regulation of 37 miRNAs out of the total 380 human miRNAs. In order to identify the cause of these miRNAs’ collapse, McKiernan et al. measured the transcript level of 4 pri-miRNAs and found no reduction of their levels, but a prominent decrease in Dicer level was identified. Dicer is an RNase III essential to the cleavage of pre-miRNA to mature miRNA. The deletion of Dicer in the neural system typically leads to neuronal dysfunction, neurodegeneration, macular degeneration, epilepsy seizure and cognitive deficits [53–57]. The loss of Dicer appears specific to TLE patients with HS and cause a failure of miRNA biogenesis, which further gives rise to degenerative changes in cortex and hippocampal morphology that have been observed in human epilepsy [54]. Aside from Dicer, other components of miRNA biogenesis may also play a role in miRNA expression regulation and may provide potential explanation for specific miRNA up-regulation. McKiernan et al. [45] also noticed that Ago2 levels were higher in non-sclerotic TLE patients. This indicates that epileptic seizures may up-regulate AGO2, by which miR-451, an up-regulated miRNA in TLE tissue, could be processed independently of Dicer [58, 59].

There are two other established miRNA regulators of note in the neural system and epilepsy: cAMP-response element binding protein (CREB) and transcription factors. CREB that regulates miR-132, contributing to the development of synaptic network plasticity. Transcription factors like myocyte enhancing factor 2 (Mef2) up-regulate the miR379-410 cluster, wherein miR-134 exhibits the essential role of promoting dendritic outgrowth of hippocampal neurons by inhibiting translation of translational repressor Pumilio2 mRNA [60, 61].

Although miRNA is predicted to regulate the expression of hundreds of proteins involved in wide-ranging biological processes, the processes regulated by miRNA may be restricted in epilepsy [23]. Inflammation and neuron morphology modulation may be two of the most prominent regulatory roles of miRNA in epileptogenesis [22]. Similar to miRNA in tumorigenesis, miRNAs exhibit a dual role in epileptogenesis– neuroprotection or induce neural cell death. A large number of studies have shown that the occurrence of epilepsy with nerve cells apoptosis, Synaptic connections reconstruction, glial fibers cell hyperplasia, abnormal pathways is closely related to the formation and inflammatory response [20, 21]. Repeated seizures can result in hippocampus neuron apoptosis in brain, the abnormal formation excitatory synapses loop, and ultimately improve the intractable temporal lobe epilepsy [23, 24]. Thus, miRNAs may be through regulating nerve cells apoptosis, synaptic contact reconstruction, glial fiber cell hyperplasia, inflammation involved in the occurrence of epilepsy development.

In the study of human disease, involves only less than 100 kinds of micrornas associated with refractory epilepsy, and collect more meaningful experimental results including up-regulation of miRNAs : miR-146a,-132,-9,-99a,-27a,-203,-135a, −4521 [62]; the down-regulation of miRNAs:miR-30a/b,-138,-324,-330,-187,-194-5p,-301a-3p,-30b-5p,-342-5p, −4446-3p [63].

Although miRNA research remains in its early stages, it certainly could have great applications in the molecular diagnosis of intractable epilepsy. As research into this area progresses, the miRNA with specificity and sensitivity will likely be found among numerous miRNAs related to epilepsy [64]. In time, this will lead to the development of effective strategies for the early screening, clinical diagnosis, treament and preventation of refractory epilepsy. The research of miRNA involved in epilepsy has great application prospect and huge potential in diagnosis and treatment of intractable epilepsy on the basis of the molecular biology.