miRNA dysregulation in traumatic brain injury and epilepsy: a systematic review to identify putative biomarkers for post-traumatic epilepsy

Traumatic brain injury (TBI) leads to post-traumatic epilepsy (PTE); hence, both TBI and PTE share various similar molecular mechanisms. MicroRNA (miRNA) is a small noncoding RNA that acts as a gene-silencing molecule. Notably, the dysregulation of miRNAs in various neurological diseases, including TBI and epilepsy, has been reported in several studies. However, studies on commonly dysregulated miRNAs and the regulation of shared pathways in both TBI and epilepsy that can identify potential biomarkers of PTE are still lacking. This systematic review covers the peer-review publications of TBI and database studies of epilepsy-dysregulated miRNAs of clinical studies. For TBI, 290 research articles were identified after screening, and 12 provided data for dysregulated miRNAs in humans. The compiled data suggest that 85 and 222 miRNAs are consecutively dysregulated in TBI and epilepsy. In both, 10 miRNAs were found to be commonly dysregulated, implying that they are potentially dysregulated miRNAs for PTE. Furthermore, the targets and involvement of each putative miRNA in different pathways were identified and evaluated. Additionally, clusters of predicted miRNAs were analyzed. Each miRNA’s regulatory role was linked with apoptosis, inflammation, and cell cycle regulation pathways. Hence, these findings provide insight for future diagnostic biomarkers.


Introduction
Epilepsy is a primeval known chronic neurological disease in individuals of all ages. The International League Against Epilepsy (ILAE), given the definition of epilepsy, is 'the recurrence of unprovoked seizures.' The current estimation of the diagnosis of epilepsy is approximately sixty million population worldwide (Fisher et al. 2005;Thurman et al. 2011). It has been estimated that 2.4 million people worldwide are diagnosed with epilepsy annually. According to the 2016 Global burden of Disease collaborators, high-income countries (HICs) have a lower prevalence of epileptic cases than low medium-income countries (LMICs) (Levira et al. 2017). This difference is probably due to various factors, such as higher numbers of head injuries and poor healthcare facilities. Head injuries are common in the acquired form of epilepsy, which is prevalent in LMICs. Head injuries can cause TBI, which can become the symptomatic form of epilepsy called PTE. PTE is the progression of seizures due to secondary brain damage due to head injury. Thus, it is essential to understand the differences between provoked and unprovoked seizures after TBI. The occurrence of seizures within 24 h of TBI is called immediate seizures; however, seizures occurring between 4 h and the first 7 days after TBI are called early post-traumatic seizures and come in the class of provoked seizures (Beghi 2003(Beghi , 2020. Reports have shown that focal hemorrhagic brain damage is the best way to determine the difference between early and late epilepsy (D'Alessandro et al. 1982;Diallo et al. 2019). Different types of head injuries further lead to post-traumatic epileptogenesis.
MicroRNAs are small noncoding RNAs that regulate gene expression by silencing and degrading target mRNAs. The mechanism of this gene silencing occurs as an extension of the synthesis of mature miRNAs. First, the generated hairpin from transcription is cleaved by drosha and Dgcr8 in the nucleus and transported to the cytosol for further cleavage to mature miRNA by Dicer (O'Brien et al. 2018). The resulting RNA duplex is a load on Argonaute protein and forms a miRNA-induced silencing complex (miRISC). Subsequently, the release of one strand of miRNA from the duplex leads to the formation of mature miRNA where the binding of miRISC with the 3' UTR takes place and further causes degradation of targeted mRNA (O'Brien et al. 2018). Since numerous reports support the crucial function of the miRNA biogenesis machinery in the brain, any disruption of this machinery leads to various neurological diseases, including epilepsy (Jimenez-Mateos and Henshall 2013; Reschke and Henshall 2015;Juźwik et al. 2019). Epileptogenesis occurs due to the dysregulation of various signaling pathways in the brain resulting from expression changes in the proteins involved.
However, the expression of these proteins is also regulated by various genetic and epigenetic switches. For example, numerous long noncoding RNAs (lncRNAs) and miR-NAs silence many genes. In recent years, miRNAs have been explored intensively in various types of epilepsy, including a number of animal models (Jimenez-Mateos and Henshall 2013; Yuan et al. 2016;Cattani et al. 2016;Tao et al. 2017). miRNAs are considered potential biomarkers in various diseases, such as cancer, gastrointestinal disease, and neurodegenerative disorders (Rupaimoole and Slack 2017;Raoof et al. 2017;Bonneau et al. 2019;Chamani et al. 2019;Ma et al. 2020). In case of PTE consists of a latent period of variable duration that can deviate from weeks to years, starting from brain injury to the occurrence of the first unprovoked seizure. Subsequently, this can lead to an array of changes in the brain, such as cell death, changes in excitatory and inhibitory neurotransmitters, axonal sprouting, and network reorganization, which further causes hyperexcitation of neurons and spontaneous seizure generation (Chang and Lowenstein 2003). In addition, the bulk displacement of brain tissue caused by mechanical trauma of the injury produces secondary responses, including changes in cerebral blood flow, intracranial pressure, and vascular permeability (Willmore 1990). Therefore, the difference in the expression of miRNAs after injury until epileptogenesis can be an interesting study to understand the regulation mechanism of various signaling pathways.
According to the National Cancer Institute, NIH, a biomarker is "A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease" (Mishra and Verma 2010). Hence, a biomarker can be used to observe the body's response to a treatment for a disease or condition. Therefore, they are also known as molecular markers and signature molecules. The Biomarkers, EndpointS, and other Tools (BEST) glossary classification of biomarkers consists of various categories (Pitkänen et al. 2021). The growing research in epilepsy biomarkers opens new doors for numerous possibilities for miRNAs as robust molecular markers (Enright et al. 2018). However, PTE is not of much concern. TBI is a significant cause of epilepsy, yet limited studies have been performed on biomarkers to distinguish between TBI and PTE. Minimal information is available about the prognosis and therapeutics of TBI. An increasing body of studies indicates that miRNAs are excellent for filling the essential need in PTE (Wang et al. 2015b, a;Ma 2017;Pietro et al. 2018;Di Pietro et al. 2018;Hanna et al. 2019;Atif and Hicks 2019). The expression of miRNAs is higher in the brain, readily crosses the blood-brain barrier and stays stable in biofluids. Though the response of miRNAs to TBI and epilepsy is complex and difficult to correlate, the indepth analysis of the involvement of physiological factors can distinguish the overlaps of TBI and PTE. Therefore, the present systematic review focused on the study of dysregulated miRNAs in TBI as well as in epilepsy to identify putative disrupted miRNAs in PTE.
Furthermore, the effect of predicted miRNAs on various signaling pathways was analyzed by identifying validated targets of miRNAs. Hence, a shared molecular signaling pathway of all PTE-associated predicted miRNAs were determined to provide insight into conserved pathways affected during post-traumatic epileptogenesis. The shared molecular pathways during PTE may offer a crucial direction toward biomarker discovery for post-traumatic epileptogenesis after head injury.

Search strategies
The miRNA expression data in epilepsy were extracted using the EpimiRBase database (https:// www. epimi rbase. eu/). The database consists of deregulating miRNAs in epilepsy data from 2007 to 2020. First, all data on miRNA expression in various forms of epilepsy were extracted in Excel format (supplementary 1). Subsequently, the miRNA expression data in humans only were filtered out. However, a systematic search was performed for TBI using the keywords "Traumatic brain injury and micro-RNA" and "TBI and miRNA" in PubMed database. The search was first conducted on 2021-06-18 and updated 2022-02-09. Therefore, articles related to miRNA dysregulation during TBI were retrieved. Hence, miRNAs that are dysregulated in TBI in humans were extracted in Excel sheet format (supplementary 2). The search strategy is also depicted in Figs. 1 and 2. Therefore, miRNAs found to be dysregulated in both epilepsy and TBI were considered PTE-associated miRNAs and further subjected to subsequent analysis. For hypothesis testing, mutual exclusive poison distribution of occurrence mutually exclusive events was used. Further, statistical testing was done to identify FDR in the selection of common miR-NAs and to understand the chances of randomly finding common miRNAs (Natarajan et al. 2012). The PRISMA flow chart systematically reviewed TBI articles from the PubMed database with differential miRNA expression. The initial library search identified 290 articles. Articles that reported miRNA dysregulation in humans only were included

Data screening
The result of the search (290 publications) was imported to the Mendeley library. The screening of the articles was conducted by the standards of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). Only primary dataset research articles were first selected. Unrelated pieces, either unrelated to miRNAs or unrelated to TBI data were removed from further consideration. Further repetitive articles on single miRNA were removed. Another filtration of reports consisting of human miRNA data was used. Therefore, TBI-dysregulated miRNAs in humans only were included. The stringent screening strategy excluded the rest of the articles (Fig. 1). Redundancy of miRNAs was removed since the present work is based on functional analysis of miRNAs and finding the function-based commonalities between TBI and Epilepsy. The identified miRNAs that are located on a common chromosome may result in the formation of clusters. This cluster functions in a coregulatory manner in various biological processes and regulates various biological signaling pathways (Chan et al. 2012). Hence, miRNA cluster analysis was performed for filtered common miRNAs in epilepsy and TBI using MetaMirClust (http:// fgfr. ibms. sinica. edu. tw/ MetaM irClu st/ MetaM irClu stSea rch. php) against the human database. It is a repository bioinformatic source of animal miRNAs that facilitates the investigation of miRNA clusters present in a particular locus.

Target genes prediction
The output miRNAs predicted to be linked with PTE were analyzed for their putative targets. Each miRNA was individually analyzed for its protein targets using the miRAnda and miRwalk web tools (http:// mirwa lk. umm. uni-heide lberg. de/). Exclusively, only experimentally validated miRNA target genes were chosen for further analysis.

Gene enrichment and pathway analysis
Each miRNA targets the expression of numerous genes that further affect various biological processes. Thus, we aimed to understand the targeted biological functions by each miRNA functional annotation using Gene Ontology (http:// geneo ntolo gy. org) of the aforesaid predicted targets of each miRNA. Additionally, pathway enrichment analysis was performed using Panther pathway analysis of Gene Ontology.

Heat map
A higher number of miRNA targets was associated with a specific pathway, indicating the degree of influence on that pathway. Thus, a heat map was prepared against the miRNAs and targets falling in the network of a particular signaling pathway. The darker color of the heat map is linked with a higher number of targets found for a specific pathway. The GO Panther pathways and miRPath web tool were used to retrieve the data of associated pathways and linked genes targeted by the miRNAs (Backes et al. 2017). R studio was used to prepare the heatmap (Hu 2021).

Gene network analysis
To understand the network of miRNA target-associated pathways. First, protein network analysis was performed using the STRING protein-protein network tool (https:// string-db. org/) with confidence of ≥ 0.4%, and a Falls discovery rate (FDR) score of ≤ 0.5 was used. Next, the predicted miRNA target list was used as input data to identify the network and probable pathways associated with the resulting network. Subsequently, a central network with at least three interactions was selected for pathway analysis.

Results
The data retrieval from EpimiRBase shows 2190 epilepsy dysregulated miRNAs in epilepsy, out of which the data of 222 miRNA expression was filtered in humans only (supplementary 1). For TBI, 290 research articles were found in the PubMed database, of which 132 were found to be relevant. Subsequently, reports that repeated the same miRNA dysregulation in a different model of TBI were excluded. Thus, 50 articles were found to be unique for each miRNA. However, only 12 articles were found to be linked with human-only miRNA dysregulation data. Thus, 85 miRNAs were found to be dysregulated in humans only after the screening of articles (Supplementary 2). These experimentally validated miRNAs were found to be dysregulated in CSF, serum, plasma, human saliva, cerebellar tissue, or any combination. Furthermore, to predict miRNAs that may be involved in PTE, overlapping miRNAs that were found to be dysregulated in both epilepsy and TBI were filtered out for further analysis (Fig. 1). Hence, in the current study, 10 miRNAs (miR-27a, miR-502, miR-130b, miR-9, miR-625, miR-660, miR-138, miR-21, miR-30a and miR-1307) were found to be common in both i.e., miRNAs dysregulated in epilepsy and TBI (P < 0.05, FDR < 0.5) ( Table 1). The miR-NAs miR-27a, miR-130b, miR-9, miR-625, miR-660, and miR-21 were found to be upregulated in both epilepsy and TBI. However, miR-502, miR-30a and miR-1307 were found to be upregulated in epilepsy but downregulated in TBI. In contrast, miR-138 was found to be downregulated in both epilepsy and TBI. Thus, these miRNAs were considered to have a possible role in PTE (Fig. 2).

miRNA-27a
miR-27a is characterized as a regulator of apoptosis, Wnt signaling and the cell cycle. Numerous studies have shown that miR-27a regulates Wnt signaling by targeting SFRP1 and FOXO1 proteins (Guo et al. 2014;Zhang et al. 2019a). Since SFRP1 positively regulates Wnt/beta-catenin, targeting SFRP1 by miR-27 negatively regulates Wnt signaling. In addition, inhibition of SFRP1 is reported to be linked to apoptosis (Guo et al. 2014). Since the regulation of Wnt signaling occurs through the binding of FOXO1 with β-catenin. Hence, by targeting FOXO1, miR-27a inhibits the binding of β-catenin with FOXO1, thereby inhibiting Wnt/β-catenin signaling (Zhang et al. 2019b). Additionally, cell cycle regulation by miR-27a is achieved by halting proliferation at the G2/M phase and promoting the differentiation of glial cells (Tripathi et al. 2019). In epilepsy, miR-27a is reported be upregulated in both animal models as well as in humans. For example, in Kainic acid-induced model, the downregulation of miR-27a suppressed the inflammatory pathway and inhibiting miR-27a mitigated seizures in rats (Lu et al. 2019).

miR-130b
miR-130b is known to regulate apoptosis by targeting PTEN Sekino et al. 2019). However, the expression of miR-130b is regulated by TNFα in tumor cells (Yang et al. 2018). miR-130b was also reported to suppress cell proliferation by regulating G2/M phase, resulting in apoptosis induction by targeting PTEN. Besides, in epilepsy, miR-130b has been reported to target mTOR, thereby regulating neuronal development and migration (Zhao et al. 2013).

miR-9
miR-9 is considered a regulator in neurogenesis and has already been observed concerning various brain pathologies. Since it is highly evolutionarily conserved, it consists of functional versatility across the evolutionary cellular context (Yuva-Aydemir et al. 2011;Coolen et al. 2012).
The association of miR-9 with several pathways makes it an interesting subject for studies in numerous diseases (Yuva-Aydemir et al. 2011;Yin et al. 2020). Moreover, miR-9 is upregulated in both epilepsy and TBI. Reports show that miR-9 regulates apoptosis signaling by inhibiting the expression of FOXO3 and SIRT1, which promotes apoptosis by inhibiting the activity of p53-and IKKβ-mediated pathways (Zhang et al. 2018(Zhang et al. , 2021. In the brain, miR-9 is reportedly involved in neurogenesis by hindering the expression of the cell cycle-promoting gene HuC (Coolen et al. 2012).

miR-625
miR-625 is a key regulator in the inflammatory pathway (Shen et al. 2019;Qian et al. 2019). Reports show that miR-625 suppresses the inflammatory response in bronchial epithelial tissue due to asthma by targeting AKT2. AKT2 triggers NF-κB signaling (Qian et al. 2019). Additionally, miR-625 has been shown to suppress NF-κB signaling by targeting other genes, such as lncRNA MALAT1 in cervical carcinoma cells and COL1A1 in human intervertebral disc degeneration (Shen et al. 2019). Moreover, miR-625 has also been reported to cease the cell cycle at G1 phase by targeting AKT2 (Qian et al. 2019).

miR-660
miR-660 regulates the cell cycle and apoptosis (He et al. 2018). Several reports suggest that miR-660 is linked to cell proliferation in various cancer cell lines. This cell cycle regulation by miR-660 occurs by targeting proteins of signaling cascades, such as SMARCA5 and AKT (Ma et al. 2020;Wu et al. 2020;Peng et al. 2020). Since AKT signaling is also known to be associated with the mTOR pathway and apoptosis, miR-660 also regulates both signaling pathways (Peng et al. 2020). In epilepsy and TBI, miR-660 is observed to be upregulated. miR-660 is present in a cluster with miR-502. Interestingly, miR-660 is upregulated in both epilepsy and TBI, while another miRNA of the same cluster showed mixed regulation.

miR-21
miR-21 is one of the most studied miRNAs. It is a key regulator in apoptosis (Krichevsky and Gabriely 2008;Sekuklu et al. 2009). Thus, the role of miR-21 in the oncogenic process has been studied in-depth. Since miR-21 is reported to target PTEN, RECK, APAF-1, STAT3, NFIB, PDCD4 and SPRY2 (Krichevsky and Gabriely 2008), reports have shown a link between miR-21 and cell proliferation and apoptosis. This miRNA has enriched expression in astrocytes of CNS. In TBI, the role of miR-21 is reported in alleviating blood-brain barrier damage and promoting neuroinflammation (Redell et al. 2011). In epilepsy, the expression of miR-21 targets STAT3 and thereby regulates neuronal death in an epileptic rat model . Similarly, in a kainic acid-induced model, miR-21 has been shown to target PTEN and thus regulate the mTOR pathway (Tang et al. 2018).

miR-502
The key observed regulation by miR-502 is in inflammation by targeting TRAF2 in chondrocyte injury (Zhang et al. 2016). In nucleus pulposus cells, miR-502 suppresses TNFα-associated apoptosis (Guo et al. 2021). Additionally, Chen et al. (2015) showed that miR-502 inhibits the cell cycle at the G1/S phase and regulates apoptosis in a hepatocellular carcinoma cell line by targeting PIK3CG.
In TBI, miR-502 is regarded as a biomarker for mild TBI, as it is downregulated in the blood of concussion patients (Davies et al. 2019). The study by Chen et al. (2020a) shows that miR-502 targets PPARγ and subsequently regulates NF-κB signaling, thereby resulting in inflammation and apoptosis of neuronal cells. In epilepsy, miR-502 is reported to be upregulated in patients (Kaalund et al. 2014). miR-502 is present in the same cluster as miR-660.

miR-30a
Research on miR-30a suggests a link between this miRNA and the regulation of the AKT signaling pathway by targeting EGFR in lung cancer cell lines (Meng et al. 2016). In liver cancer, miR-30a is reported to target DNMT3a (Chen et al. 2020b). Thus, as part of AKT signaling, miR-30a regulates apoptosis in HepG2 cells (Meng et al. 2016). In addition, downregulation of miR-30a promoted cell proliferation by regulating Wnt signaling in squamous cell carcinoma (Qi et al. 2017). In TBI, upregulated miR-30a has been shown to regulate BDNF in cortical injury rats (Tan et al. 2020). Interestingly, miR-30a was reported to be downregulated in epilepsy (McKiernan et al. 2012).

miR-1307
Reports on miR-1307 show that downregulated miR-1307 promotes cell proliferation by targeting FOXO3A in prostate cancer (Qiu and Dou 2017). However, in breast cancer, miR-1307 is reported to target SMYD4 and contribute to development in cell lines (Han et al. 2019). In hepatocellular carcinoma, miR-1307 is observed to promote tumorigenesis and metastasis by targeting DAB2IP (Chen et al. 2019). In TBI, miR-1307 was reported to be dysregulated in human saliva after prolonged concussion (Johnson et al. 2018). Whereas in epilepsy, miR-1307 is downregulated in temporal lobe epilepsy (Johnson et al. 2018). The analysis of miRNA clusters showed the presence of various clusters in 10 predicted miRNAs (Table 2). miR-27a, miR-502, miR-130b, miR-9, and miR-660 are present in chromosomes with other miRNAs and possess an individual cluster. Besides, miR-502 and miR-660 were found to be present in a single cluster and might be co-expressed. However, no cluster was found for miR-625, miR-138, miR-21, miR-30a and miR-1307 in MetaMirClust.
Using the 10 identified miRNAs, the targets were predicted individually. Each miRNA shows numerous targets. Thus, 1014 experimentally validated target genes were found for miR-27a. Similarly, 252, 1322, 731, 511, 74, 501, 735, 657 and 634 experimental validated targets were consecutively identified for miR-27a, miR-502, miR-130, miR-9, miR-625, miR-660, miR-138, miR-21, miR-30a and miR-1307. Since miRNAs regulate the expression of their target genes through the gene silencing mechanism. The targets of each miRNA were also analyzed for gene enrichment and pathways. The pathways associated with already predicted targets were analyzed using GO Panther pathways. The 10 microRNAs were linked with the pathways in Table 1. Since dysregulation of all 10 predicted miRNAs simultaneously affects a natural biological system. Hence, a combined analysis of regulated pathways was also performed using the miRPath web tool. The output data show that miRNAs are primarily involved in the regulation of secretory pathways (Table 4) (p < 0.001, FDR < 0.005). Similarly, miRNAs are observed to be involved in histone acetylation, lysine acetylation, oxidoreductase activity and regulation of steroid hormone receptor signaling (Table 3). The molecular function analysis shows the target gene involved, where 145 target genes of miRNAs are associated with binding function, 101 genes are linked with catalytic activity, and 63 genes function as regulators (Table 4).
Since every miRNA consists of numerous targets associated with a particular pathway, the created heat map depicted the pathways that are majorly affected by each miRNA (Fig. 3). miR-21 displayed an association with pathways that are linked with epilepsy according to the literature. Similarly, miR-30a, miR-130b and miR-9a showed the most targets related to pathways such as the apoptosis, NF-κB and p53 signaling pathways. STRING gene network analysis results also demonstrated the targets of predicted miRNAs in general and related pathways associated with PTE (Fig. 4). The central network depicted the presence of targets linked with pathways such as apoptosis, cancer, and cell cycle regulation, which is similar to the results of miR-Path and the heat map.

Discussion
An increasing number of miRNA studies and clinical trial reports have shown that miRNAs are used as biomarkers in numerous disorders and diseases (Bonneau et al. 2019;Hanna et al. 2019). However, the requirement of clinical and preclinical research on miRNAs in epilepsy still needs to be forged. Similarly, lacunae remain in vivo and in vitro studies focusing on identifying miRNAs in PTE. Hence, in this study, using in silico tools, we focused on providing insight into miRNAs that can be involved in PTE. Here, we identified miR-27a, miR-502, miR-130b, miR-9, miR-625, miR-660, miR-138, miR-21, miR-30a and miR-1307 as dysregulated in clinical studies of both TBI and epilepsy. Interestingly, miRNAs which are upregulated in one condition and downregulated in other could be due to various biological factors such as different biological samples (e.g., serum or CSF) or analyzed microRNAs in cohorts with different TBI severity of injury or seizure frequency or severity score. Therefore, miRNA that are commonly dysregulated in both TBI and epilepsy were chosen for further analysis regardless of positive or negative levels. However, regulation of selected miRNAs was further verified in their particular studies, including animal studies (Supplementary Table 1). As most of the miRNAs are conserved in nature, their regulation in the disease model is a concrete way to assess the expression pattern in actual human disease. The regulation of selected miRNAs was found to be mostly concordant for selected ten miRNAs except for miR-138, which was found upregulated in mouse brain profiling for epilepsy and without a clear understanding of the direction of regulation in TBI. This could be due to experimental or RNA sample handling errors. Nonetheless, further clinical/human studies are needed to reach any inference for miRNAs regulation and its regulation of apoptosis in Epileptogenesis from TBI. One of the key findings of this review is the identification of commonly upregulated and downregulated miRNAs in TBI as well as in epilepsy. Therefore, this suggests a shared mechanism of regulation of pathways in both TBI and epilepsy. Since PTE is the progression of epilepsy after TBI, the predominant miRNAs hint at a link between the molecular mechanism and share signaling pathways of epileptogenesis after TBI. Therefore, the functional analysis by Gene Ontology and KEGG pathways showed that cancer pathways were involved most individually with all miRNAs. Evidence also suggests the link between cancer and epilepsy as the probability of seizures increases during cancer (Singh et al. 2007;Adelöw et al. 2006). Hence pathways related such as the cell cycle, NF-κB, p53 and apoptosis were also found to be significantly affected by the targets of the predicted miRNAs individually. For example, targets of miR-130b, miR-21 and miR-30 are commonly involved in the p53 pathway. Since numerous studies have already reported the involvement of the p53 gene and apoptosis pathway in the pathogenesis of PTE and neurodegeneration, these pathways may be affected by the role of such predicted miRNAs in the regulation of epileptogenesis (Engel et al. 2007(Engel et al. , 2010. Similarly, the NF-κB pathway consists of gene targets of miR-502 and  miR-9. The inflammation pathway is one of the key pathways involved in the pathogenesis of PTE. Thus, the targets of both miRNAs might be involved in the regulation of the pathogenesis of PTE. Additionally, the summary of shared pathways regulated by miRNAs in PTE shows that central apoptosis and inflammation are major signaling pathways that are affected by both TBI and epilepsy (Fig. 5). This finding concurs with previous PTE studies that suggest that apoptosis-associated neuronal death occurs post-inflammation Sun et al. 2021). Similarly, all the miRNA combinedly targets various pathways related to epilepsy such as apoptosis, inflammation, and neurodegeneration. For example, in Table 3 nitric oxide synthase regulation is associated with neurodegeneration, a subsequent product of both epileptic seizures and TBI (Tewari et al. 2021). The present data also showed that Oxidoreductase is targeted by miRNAs which is in line with observations about link of Oxidoreductase with epilepsy in the Japan region (Saruwatari et al. 2014). Thus, this signal transduction cascadebased evidence suggests the link of identified miRNAs with epilepsy and TBI. Furthermore, reports show that epileptic seizures enhance the process of hippocampus adult neurogenesis. This neurogenesis is induced by activation of a number of signaling pathways. For example, a recent study shows that Wnt/β catenin pathways is directly linked with epilepsy associated adult neurogenesis (Chen et al. 2020c). Additionally, neuroinflammatory pathways of Cox-2, which are linked with proteoglycans and prostaglandins, also facilitate adult neurogenesis (Barreiro-Iglesias 2021; Mencio et al. 2021). The present study shows that common miRNAs regulate genes related to these pathways. For example, miR-9 is reported as a direct neurogenesis regulator. Whereas miRNAs 27a, 502, 138, and 21 found enriching proteoglycan pathways lined with Cox -2 mediated neuroinflammation.
One of the significant characteristics of epilepsy is electrophysiologically hyperexcited neurons of the neocortex and hippocampus, particularly the CA3 region. Such hyperexcitation leads to High-Frequency Oscillation (HFO) electrophysiology. Since neuron excitation is a function of channel proteins, the occurrence of HFOs is directly attributed with various kind of channel proteins e.g., Navs, AMPAR, GABAR, and Kvs. Reports show that channel proteins e.g. Navs, Kvs AMPAR, and NMDAR, are regulated by many miRNAs (Gross and Tiwari 2018). However, only miR-9 directly targets and regulates expression of Nav1.1 and Nav 1.2 (Sun et al. 2015). In addition, evidence shows that neuroinflammation is linked with HFO where present study has Fig. 3 Heat map summary of each miRNA commonly dysregulated in TBI and epilepsy. Their associated pathway suggests the probable role of miRNAs in PTE. The darker color shows the involvement of a higher number of each miRNA targets in a particular pathway shown the relation of neuroinflamtion pathway regulation by common miRNAs (Sun et al. 2022).
The classification of biomarkers is performed between diagnostic, prognostic, predictive, and therapeutic biomarkers (Pitkänen et al. 2021). However, the current review focuses only on prognostic and therapeutic biomarkers for PTE. Several biomarkers are under investigation for TBI and epilepsy. For example, GFAP, S100B, IL-6, HMBG1, and UCH-L1 are considered biomarkers in both TBI and epilepsy. Among all, GFAP is considered as one of the biomarkers for PTE (Liliang et al. 2010;Okonkwo et al. 2013;Çevik et al. 2019;Pitkänen et al. 2021). Additionally, the US FDA recently approved GFAP and UCH-L1 as biomarkers for mild TBI (Pitkänen et al. 2021). Likewise, numerous studies on genetic biomarkers have also been published (Pitkänen et al. 2021). However, both genetic and protein biomarkers fail to provide prior information on the probability of PTE after brain insult. Additionally, molecular biomarkers are highly stable in the circulatory environment of the human body (Sanz-Rubio et al. 2018). In addition, an earlier diagnosis can provide better Here, miRNAs may provide a crucial opportunity for the earlier diagnosis of diseases, including PTE. An increasing body of evidence suggests that numerous miRNAs are considered biomarker candidates for post-traumatic epilepsy. For example, miR-106b, miR-130a, miR-301a, miR-30b, let-7d, miR-194, miR-4521, and miR-134 can be biomarkers of epilepsy (Wang et al. 2015b, a;An et al. 2016) and may be probable biomarkers of PTE (Ma 2017). Current study also shows that these 10 intersected expressing miRNAs between TBI and epilepsy are also involved in regulation of conventionally known protein biomarkers (Table 5). Thus, it is necessary to uncover the field of biomarkers for PTE that can provide prior and predictive information about the response to therapy and ultimately improve the therapeutics overall against PTE.
In the current review, miRNAs such as miR-21, miR-9 and miR-130b were found to be upregulated. miR-21 shows upregulation in the initial period of head injury from 6 h. until the late period after 72 h. (Raoof et al. 2017;Pitkänen et al. 2021). It was also found to be upregulated in epilepsy. As a prognostic biomarker consists of properties of showing quantitative changes in the body fluid or tissues before the onset of diseases. Since miRNAs are present in vesicles known as exosomes that are released from tissue to body fluids after crossing BBB (Petrescu et al. 2019). It provides information about the predisposed condition of a specific disorder that might turn into a disease. Hence, we predicted that it may be a probable candidate for further study as a prognostic biomarker of PTE. Importantly, miR-21 is a primary target of the apoptosis pathway. Thus, miRNAs that share the same signaling axis might be further studied as candidate prognostic biomarkers. For example, miR-9 and miR-130 can also be molecules of interest for further analysis as prognostic biomarkers.
Therapeutic markers are molecules that drugs can target to treat various diseases. Generally, proteins are considered good therapeutic markers. In epilepsy, HMGB1 has been contemplated as a potential therapeutic biomarker. As a result, it can differentiate patients with a higher risk of epilepsy (Au et al. 2012;Zhu et al. 2018;Kobylarek et al. 2019). The current review attempts to understand the link between miRNAs and PTE as therapeutic biomarkers. Studies suggest that miR-27a, miR-21 and miR-30b are considered therapeutic markers for epilepsy (Raoof et al. 2018;Venø et al. 2020). Additionally, preclinical data shows that miR-21 mimic can suppress seizures (Tang et al. 2018). Our data are also in concordance with those of previous studies. Hence, in-depth in vitro and in vivo studies are required to draw concrete  conclusions about therapeutic markers of PTE. However, our review provides a narrowed direction for miRNA, which should be considered for further studies in PTE.
In summary, we focused on miRNA candidates with commonalities in TBI and epilepsy to provide insight into the progression of epileptogenesis and identified ten miRNAs. Thus, we considered these 10 miRNAs linked with PTE. Additionally, apoptosis, inflammation, NFκB and cell cycle regulation pathways were found to be major targets of miR-NAs. Furthermore, we noted the shared regulatory pathway of 10 identified miRNAs with putative links with PTE. Although we have some hints about potential miRNAs associated with PTE, future miRNA research must understand the correlation of the mechanism in PTE while addressing various factors, such as age and severity of the injury.