Assessment of expression of calcium signaling related lncRNAs in epilepsy

Calcium signaling is a metabolic pathway that is essential in neurons development and can be involved in the pathobiology of epilepsy. We assessed expression of three mRNA coding gene (SLC1A1, SLC25A12, and ATP2B2) and three related long non-coding RNAs (LINC01231:1, lnc-SLC25A12-8:1 and lnc-MTR-1:1) from this pathway in 39 patients with refractory epilepsy and 71 healthy controls. Expression of all genes except for lnc-SLC25A12 was higher in total epileptic cases compared with controls (P values = 0.0002, < 0.0001, < 0.0001, 0.049 and 0.0005 for SLC1A1, SLC25A12, LINC01231, ATP2B2 and lnc-MTR-1, respectively. When we separately compared expression of genes among males and females, SLC1A1, SLC25A12, LINC01231 and lnc-MTR-1 showed up-regulation in male cases compared with male controls. Moreover, expressions of SLC1A1 and SLC25A12 were higher in female cases compared with female controls. Remarkably, SLC25A12 was found to have the highest sensitivity value (= 1) for differentiation of epileptic cases from controls. Moreover, lnc-MTR-1 and lnc-SLC25A12 were sensitive markers for such purpose (sensitivity values = 0.89 and 0.87, respectively). The highest value belonged to LINC01231 with the value of 0.76. Taken together, this study demonstrates dysregulation of calcium-signaling related genes in epileptic patients and suggests these genes as potential biomarkers for epilepsy.

The role of calcium signaling related genes has been discussed in the context of brain disorders such as autism spectrum disorder 15 .Since calcium signaling has an efficient role in the neurodevelopmental disorders through regulation of normal neuronal activity, the related genes may play a role in the epilepsy as well.Thus, we analyzed expression of SLC1A1, SLC25A12, and ATP2B2 genes that encode calcium signaling proteins that play a part in storing, regulation, and transmission of calcium in neurons.We also assessed expression of three lncRNAs that are related with these genes, namely LINC01231:1, lnc-SLC25A12-8:1 and lnc-MTR-1:1T, respectively, as predicted by the LNCipedia 16 and RNAcentral 17 databases.The regulatory network constructed by lncRNA-mRNA interactions may affect epileptogenesis and can be considered as a putative target for therapeutic interventions, particularly in refractory epilepsy.

Patients and controls
In total, 40 patients with refractory seizures and 71 healthy persons (41 males and 30 females) were enrolled in the current study.Patients were classified according to the definition of International League Against Epilepsy (ILAE), "failure of adequate trials of two tolerated, properly selected and used antiepileptic drug plans (whether as monotherapy or in combination) to accomplish continued seizure freedom" 18 .Diagnosis was based on electroencephalogram and brain magnetic resonance imaging.Informed consent forms were signed by all participants.The study protocol was permitted by ethical committee of Shahid Beheshti University of Medical Sciences.

Experiments
In this assay, we measured expression of six genes (coding and non-coding) which are shown in Table 1.For this purpose, we took blood samples from 39 refractory epileptic patients (5 cc of whole blood).Total RNA extraction and cDNA synthesis were accomplished by using RiboEx and SMOBIO kits, respectively.Then, real time PCR was performed in the ABI system using specific designed primers.

Statistical analysis
Statistical analysis was accomplished in GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA, USA).Expression levels of three genes encoding ion channels and transporters, namely SLC1A1, SLC25A12, ATP2B2 and their related lncRNAs, namely LINC01231, lnc-SLC25A12, and lnc-MTR-1 were measured in the peripheral blood obtained from 39 patients with refractory epilepsy and 71 healthy controls.Expression levels were assessed using the comparative − delta Ct method.The normal/gaussian distribution of the values was evaluated using the Shapiro-Wilk test.Unpaired t test or non-parametric test (Mann-Whitney U test) was used to identify differentially expressed genes between two groups.Effects of main factors on gene expression were determined using the two-way ANOVA and Tukey post hoc tests.Correlations between expression levels of genes were measured using the Spearman's rank correlation coefficient.

Ethics approval and consent to participant
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent forms were obtained from all study participants.The study protocol was approved by the ethical committee of Shahid Beheshti University of Medical Sciences.All methods were performed in accordance with the relevant guidelines and regulations.

General data
The study included 39 patients with refractory epilepsy (16 male subjects and 23 female subjects).Moreover, we included 71 healthy persons as controls (41 male subjects and 30 female subjects).

Expression assays
Expression levels of SLC1A1, SLC25A12, ATP2B2 and their related lncRNAs were significantly different between patients with epilepsy and healthy subjects (Fig. 1).www.nature.com/scientificreports/Group factor (disease) had a significant effect on expression levels of SLC1A1, SLC25A12, LINC01231 and lnc-MTR-1.On the other hand, gender factor and interaction between gender and group had no effect on expression levels of any of genes (Table 2); so, we did not perform post hoc tests for multiple comparisons.
Expression of all genes except for lnc-SLC25A12 was higher in total epileptic cases compared with controls (P values = 0.0002, < 0.0001, < 0.0001, 0.049 and 0.0005 for SLC1A1, SLC25A12, LINC01231, ATP2B2 and lnc-MTR-1, respectively (Table 3).When we separately compared expression of genes among males and females, SLC1A1, SLC25A12, LINC01231 and lnc-MTR-1 showed up-regulation in male cases compared with male controls.Moreover, expressions of SLC1A1 and SLC25A12 were higher in female cases compared with female controls.
Since most of Ct values were not normally distributed, we used non parametric Spearman's correlations between RNA expression levels among the epileptic patients and healthy controls.When analyzing the correlation between expression levels of genes, we noticed that the most robust correlations were between lnc-MTR-1/ ATP2B2, ATP2B2/lnc-SLC25A12 and lnc-MTR-1/lnc-SLC25A12 pairs among epileptic cases with correlation coefficients of 0.73, 0.73 and 0.68, respectively (Table 4).
Remarkably, SLC25A12 was found to have the highest sensitivity value (= 1) for differentiation of epileptic cases from controls.Moreover, lnc-MTR-1 and lnc-SLC25A12 were sensitive markers for such purpose (sensitivity values = 0.89 and 0.87, respectively).The highest value belonged to LINC01231 with the value of 0.76 (Table 5).
Table 2. Graphpad prism output from analysis of effect of group and gender (tests of between-subjects effects) on expression of three genes encoding ion channels and transporters including SLC1A1, SLC25A12, ATP2B2 and their related long non-coding RNAs, namely LINC01231, lnc-SLC25A12, and lnc-MTR-1 in total epileptic patients compared to healthy controls.Two-way ANOVA and Tukey post hoc tests were used to analyze the effects of main factors (disease and gender) and their interaction on expression levels (*P value < 0.05, **P value < 0.01, ***P value < 0.001 and ****P value < 0.0001). 1 Sum of Squares. 2 F of Variance.Significant values are in bold.

Group effect
Gender effect Interactions SS

Discussion
Calcium signaling has an important role in the neurological disorders.In fact, the triad of excitotoxicity, calcium and mitochondria has been found to be involved in synaptic neurodegeneration 19 .Malfunction of proteins related with this pathway is followed by mitochondrial calcium toxicity and excitotoxic dendritic loss and hence neurodegeneration 19 .Meanwhile, neurons channel proteins and synaptic plasticity are two targets of lncRNAs which are intertwined with epilepsy pathogenesis 20 .
It is also confirmed that astrocytes have a key role in controlling the conformation of the extracellular fluids, and can directly cooperate with neurons by discharging gliotransmitters.Notably, astrocytic intracellular Calcium signals increase discharge of signaling elements, either via synaptic or non-synaptic routes.Thus, astrocytic calcium signals have crucial roles in epileptogenesis 21 .Calbindin-D28K, a member of calcium binding protein family has been found to be down-regulated in the cortical tubers of patients with tuberous sclerosis complex associated with refractory epilepsy 22 .However, expression of other calcium-related proteins has not been assessed in refractory versus non-refractory epilepsy.
In the current study, we measured expression of a number of mRNA coding genes and lncRNAs in epileptic patients.Expression of all genes except for lnc-SLC25A12 was higher in total epileptic cases compared with controls.When we separately compared expression of genes among males and females, SLC1A1, SLC25A12, LINC01231 and lnc-MTR-1 showed up-regulation in male cases compared with male controls.Moreover, expressions of SLC1A1 and SLC25A12 were higher in female cases compared with female controls.A previous study has reported significant elevation of SLC1A1 expression in dentate granule cells from rats with spontaneous seizure compared with similar cells from control rats 23 .Moreover, expression of these transcripts have been shown to be high in human dentate granule cells from patients with temporal lobe epilepsy and as well as in dysplastic neurons in cortical dysplasia compared with non-dysplastic neurons of control tissues obtained from autopsy 23 .Thus, the results of our study provide further evidence for involvement of SLC1A1 in the pathobiology of epilepsy.We also detected up-regulation of LINC01231, the lncRNA that interacts with the SLC1A1 mRNA.This lncRNA   www.nature.com/scientificreports/has also been shown to be upregulated in major depressive disorder cases and down-regulated in Alzheimer's disease patients 24 .Thus, it is involved in a variety of neurologic disorders.
In line with our findings, mutations in SLC25A12 gene are associated with infantile epileptic encephalopathy, psychomotor retardation, hypomyelination of the CNS and seizures 25 .Moreover, two polymorphisms within SLC25A12 gene (rs2292813 and rs2056202) are associated with autism spectrum disorder 26 .In spite of upregulation of this gene in epileptic cases, expression of the related lncRNA with this gene (lnc-SLC25A12) was not different between cases and controls.This finding might suggest that the role of this mRNA coding gene in epilepsy is independent from the related lncRNA.
We also noticed that the most robust correlations were between lnc-MTR-1/ATP2B2, ATP2B2/lnc-SLC25A12 and lnc-MTR-1/lnc-SLC25A12 pairs among epileptic cases.In fact, the strongest correlation was detected between ATP2B2 and its related lncRNA, i.e. lnc-MTR-1.However, the correlations between other mRNA coding genes and their related lncRNAs were not so strong.
Remarkably, SLC25A12, lnc-MTR-1 and lnc-SLC25A12 were found to be sensitive markers for differentiation of epileptic cases from controls.However, the specificity of these transcripts were not appropriate.Taken together, this study demonstrates dysregulation of calcium-signaling related genes in epileptic patients and suggests these genes as potential biomarkers for epilepsy.

Figure 2 .
Figure 2. ROC curves of six studied genes in patients with epilepsy disease.

Table 3 .
Expression ratio (fold change) of three genes encoding ion channels and transporters and their related long non-coding RNAs, namely LINC01231, lnc-SLC25A12, and lnc-MTR-1 in epileptic patients compared to healthy controls.Significant values are in bold.

Table 5 .
ROC curve analysis for transcript levels of six studied genes in patients with epilepsy.