Aberrant Expression of Serum MicroRNA-153 and -199a in Generalized Epilepsy and its Correlation with Drug Resistance

Abstract

Background

Epilepsy is one of the common neurological disorders affecting approximately 50 million people worldwide. Despite the recent introduction of new antiepileptic drugs, about one-third of patients with epilepsy have seizures refractory to pharmacotherapy. Early recognition of patients with drug-resistant epilepsy may help direct these patients to appropriate nonpharmacological treatment.

Purpose

The possible use of serum microRNAs (miRNAs) as noninvasive biomarkers has been explored in various brain diseases, including epilepsy. In this study, we are aiming at analyzing the expression levels of circulating miRNA-153 and miRNA-199a in patients with generalized epilepsy and their correlation with drug resistance.

Methods

Our study comprised 40 patients with generalized epilepsy and 20 healthy controls. 22 patients were drug-resistant and 18 patients were drug-responsive. The expression levels of miRNA-153 and -199a in serum were analyzed using quantitative real-time polymerase chain reaction. Data analysis was done by IBM SPSS Statistics 20.0.

Results

The expression of miRNA-153 and -199a in serum was significantly downregulated in patients with generalized epilepsy compared with that of the healthy control (P < .001). Combined expression level of serum miRNA-153 and -199a had a sensitivity of 85% and a specificity of 90% in the diagnosis of generalized epilepsy. Furthermore, the expression levels of miRNA-153 and -199a were significantly decreased in drug-resistant patients compared to the drug-responsive group, and the combination of both markers gave the best results in differentiating between the two groups.

Conclusion

We suggest that serum miRNAs-153 and -199a expression levels could be potential noninvasive biomarkers supporting the diagnosis of generalized epilepsy. Moreover, they could be used for the early detection of refractory generalized epilepsy.

Introduction

Epilepsy is a group of chronic neurological disorders characterized by spontaneous, recurrent, and unpredictable seizures. It is one of the most common central nervous system diseases affecting people of all ages, social classes, races, and geographical regions. It affects approximately 50 million individuals worldwide. Around 80% of epilepsy cases occur in the developing countries, and three-fourths of the affected individuals do not get appropriate treatment. ¹ Current interventions and antiepileptic drugs (AEDs) are ineffective in more than 30% of the patients. The pathogenic mechanisms underlying drug-resistant epilepsy remain unknown. ²

Conclusive diagnosis of refractory epilepsy always requires a long treatment cycle of several years, during which, recurrent seizures may not only aggravate patients’ cerebral function but also lead to delays in terms of the optimal opportunity for comprehensive treatment, including surgery. Besides, refractory epilepsy creates a notable threat to patients’ families and even society, whether economically, physically, or psychologically. Thus, researchers are aimed at identifying a rapid, noninvasive, accessible, and inexpensive method for the early identification of refractory epilepsy.^{3, 4}

MicroRNAs (miRNAs) are a group of small noncoding RNAs with an average length of 22 nucleotides that negatively regulate specific gene expression, mainly through interaction with target mRNAs, leading to their degradation or inhibiting their translation resulting in overall lower protein levels in cells. ⁵ In recent years, the role of miRNAs in the pathogenesis of epilepsy is a fast-expanding area of research.^{6, 7}

Previous studies have demonstrated the role of two miRNAs, miRNA-153 and -199a, in drug-resistant focal epilepsy patients;^8–10 however, to the best of our knowledge, their levels in generalized epilepsy and their correlation with drug resistance in this group have not been assessed until now.

Methods

Patients and Control Group

All the patients were recruited from the outpatients of epilepsy clinic at the Neurology Department, Alexandria University Hospital from August 2019 to February 2020. The control subjects were recruited from the medical biochemistry department staff members, Alexandria University, and were confirmed healthy and neurologically normal by medical history and general examinations, and had no history of seizures or exposure to AEDs. Major exclusion criteria were a history of autoimmune diseases, allergic response, immune deficiency disorder, diabetes, stroke, heart disease, atherosclerosis, malignancy, or a systemic or central nervous system infection two weeks before sample collection. Informed consent to participate in this study was obtained from each subject. The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Local Ethics Committee of the Faculty of Medicine, University of Alexandria.

Drug-resistant epilepsy, diagnosed according to the guidelines for the classification and diagnosis of epilepsy of the International League Against Epilepsy, was defined as the 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. ¹¹ In our study, all the patients with refractory epilepsy were still on medications at the time of sampling. Drug-responsive epilepsy was defined as being seizure-free for at least 1 year.

Blood Sample Collection

Up to 3 mL whole blood was collected from each participant and was processed for serum isolation within 3 h of collection by centrifugation 1200 × g for 10 min to 15 min. The collected serum samples were stored at −80°C and were not thawed until use. Hemolyzed serum samples were excluded.

Extraction of Total RNA

Total RNA isolation from serum samples was carried out with the Qiagen miRNeasy Mini Kit (Qiagen, CA, USA) according to the manufacturer’s instructions. Around 5 µL synthetic Caenorhabditis elegans miRNA cel-miR-39 (5′-UCAC­CGGGUGUAAAUCAGCUUG-3, 10 nM) was used as spike-in control and added directly to each sample.

Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Quantification of miRNA-153 and -199a expression using the TaqMan miRNA assays was done using two-step RT-PCR:

1. Reverse transcription (RT) step: cDNA was reverse transcribed from purified RNA samples using specific miRNA stem-loop primers from the TaqMan miRNA assays and reagents from the TaqMan miRNA Reverse Transcription Kit (Applied biosystem, MA, USA).

2. Quantitative real-time PCR (qPCR) step: PCR products were amplified from cDNA samples using the TaqMan miRNA assays together with the TaqMan Universal PCR Master Mix (Applied biosystem, MA, USA). Thermocycling was done using Applied Biosystems StepOne Real-Time PCR System. The relative expression was calculated using the 2^−ΔΔCT method.

Statistical Analysis of the Data

Data were analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp.). We used Kolmogorov–Smirnov test to assess the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, standard deviation, median, and interquartile range (IQR). Obtained results’ significance was judged at the 5% level. Because the expression levels of serum miRNA-153 and -199a were not normally distributed, their group differences were analyzed by a nonparametric test. Mann–Whitney U test was used. Receiver operating characteristic (ROC) curves were done to assess the diagnostic value by calculating the area under the curve (AUC), sensitivity, and specificity.

Results

Characteristics of the Study Population

We enrolled 40 patients with generalized epilepsy and 20 healthy controls in our study. The mean age of patients was 31.6 ± 6.83 years old. Generalized epilepsy group included 28 males and 12 females, while the control group comprised 12 males and 8 females. Among 40 recruited patients, 22 were diagnosed with drug-resistant epilepsy. There was no significant difference in age or gender ratio between the patient and control groups (P > .05).

Differential Expression of miRNA-153 and -199a in Serum Between Generalized Epilepsy Patients and Healthy Controls

Our data revealed that both miRNA-153 and miRNA-199a are significantly downregulated in the generalized epilepsy group as compared with controls (P < .001; Table 1, Figure 1).

Table 1. Comparison Between the Control Group and Generalized Epilepsy Patients According to miRNA-153 and -199a.

–	Generalized (n = 40)	Control (n = 20)	U	P
MicroRNA-153	–	–	–	–
Mean ± SD	0.5 ± 0.2	1.2 ± 0.7	124.0*	<.001*
Median (Minimum–Maximum)	0.4 (0.2–0.9)	1.1 (0.3–2.5)
MicroRNA-199	–	–	–	–
Mean ± SD.	0.4 ± 0.3	1.5 ± 1.3	132.0*	<.001*
Median (Minimum–Maximum)	0.3 (0.1–1.1)	1.1 (0.2–4.2)

Note: Data were expressed by median (minimum–maximum); U, Mann–Whitney test; P, P value for comparing between the two studied groups; * statistically significant at P ≤ .05.

Figure 1. Comparison Between the Control Group and Generalized Epilepsy Patients According to miRNA-153 and -199a.

The sensitivity and specificity of serum miRNAs-153 and -199a expression levels as markers for diagnosing generalized epilepsy have been determined by plotting the ROC curve. At the cutoff value of 0.64, the sensitivity of serum miRNA-153 in detecting generalized epilepsy was 75% and its specificity was 70%, while miRNA-199a had a sensitivity of 85% and a specificity of 80% at the cutoff value of 0.529 for the diagnosis of generalized epilepsy. The combined expression level of serum miRNA-153 and -199a had a sensitivity of 85% and a specificity of 90% (Figure 2, Table 2).

Figure 2. ROC Curve for miRNA-153, miRNA-199a and Their Combination to Discriminate Generalized Patients (n = 40) from Control Group (n = 20).

Table 2. Validity (AUC, Sensitivity, Specificity) for miRNA-153, miRNA-199a and Their Combination to Diagnose Generalized Epilepsy.

–	AUC	P	95% CI	Cut off	Sensitivity	Specificity	PPV	NPV
miRNA-153	0.845	<.001*	0.738, 0.952	≤0.64	75.0	70.0	83.3	58.3
miRNA-199a	0.835	<.001*	0.715, 0.955	≤0.529	85.0	80.0	89.5	72.7
miRNA-153 and -199a	0.875	<.001*	0.769, 0.981	–	85.00	90.00	94.4	75.0

Abbreviations: AUC, area under a curve; P value, probability value; CI, confidence intervals; NPV, negative predictive value; PPV, positive predictive value.

Note: * statistically significant at P ≤ .05.

Serum levels of miRNAs-153 and -199a are differentially expressed in drug-resistant patients compared with those in drug-responsive patients.

Serum miRNA-153 and -199a expression levels were downregulated in drug-resistant patients compared to drug-responders (Table 3).

Table 3. Comparison Between Nonresponder Patients and Drug Responders According to Serum Expression Levels of miRNA-153 and -199a in Generalized Group (n = 40).

–	Nonresponder (n = 22)	Responder (n = 18)	U	P
MicroRNA-153	–	–	–	–
Mean ± SD	0.31±0.11	0.65±0.17	24.0*	<.001*
Median (Minimum–Maximum)	0.27 (0.19–0.53)	0.66 (0.31–0.90)
MicroRNA-199a	–	–	–	–
Mean ± SD	0.26 ± 0.14	0.49± 0.37	134.0	0.08
Median (Minimum–Maximum)	0.21 (0.06–0.53)	0.33 (0.12–1.15)

Note: U, Mann–Whitney test; P, P value for comparing between nonresponder and responder; * statistically significant at P ≤ .05.

miRNA-153 at the cutoff value of 0.37 can predict drug response with a sensitivity 88.89%, where expression level >0.37 predicts drug response and ≤0.37 predicts drug resistance. ROC analysis showed that the accuracy for the identification of patients with drug-resistant generalized epilepsy using miRNA-153 expression level was AUC = 0.939, and AUC = 0.662 in the case of miRNA-199a. However, the combination of both miRNA-153 and -199a yielded AUC =1 with 100% sensitivity and specificity (Figure 3, Table 4).

Figure 3. ROC Curve for miRNA-153, miRNA-199a and Their Combination to Predict Responders in Generalized Epilepsy Group.

Table 4. Validity (AUC, Sensitivity, Specificity) for miRNA-153 and -199a to Predict Responders in Generalized Epilepsy Group.

–	AUC	P	95% C.I	Cut off#	Sensitivity	Specificity	PPV	NPV
miRNA-153	0.939	<.001*	0.870, 1.009	>0.37	88.89	81.82	80.0	90.0
miRNA-199a	0.662	0.08	0.485, 0.838	>0.247	66.67	63.64	60.0	70.0
miRNA-153 and -199a	1.000	<.001*	1.000, 1.000	–	100.0	100.0	100.0	100.0

Abbreviations: AUC, area under a curve; P value, probability value; CI, confidence intervals; NPV, negative predictive value; PPV, positive predictive value.

Note: * statistically significant at P ≤ .05.

Discussion

To date, no generally accepted biological or molecular markers for drug responsiveness to conventional AEDs exist. ¹² Nervous system cells can secrete miRNAs into the circulation and miRNAs are stable in blood, which makes miRNAs potential candidates as noninvasive biomarkers for early detection of drug-resistant epilepsy. Studies on refractory epilepsy have offered the exciting possibility of miRNAs acting as potential biomarkers for epilepsy diagnosis, such as miRNA-134, miRNA-129-2-3p, and miRNA-935 in plasma with high sensitivity and specificity.^{13, 14} In addition to acting as a biomarker, miRNAs reduce target mRNA stability and translation, inhibiting the expression of multiple proteins and could therefore provide clues to disease etiology and pathogenesis of drug resistance. ⁶

It is more realistic to assume that no single biomarker will achieve 100% sensitivity and specificity and that a combination of various biomarkers together with clinical data is more likely to be used in clinical practice. Our study indicated a significant downregulation of both miRNAs-153 and -199a in the serum of patients with generalized epilepsy. In addition, we showed the possible use of miRNAs-153 and -199a in the diagnosis of generalized epilepsy. The combination of both miRNAs showed the best diagnostic value for generalized epilepsy with 85% sensitivity and 90% specificity.

Another finding of our study was the possible use of these miRNAs in combination for the prediction of drug-resistance in patients with generalized epilepsy with 100% sensitivity and specificity.

Similar results were reported by studies in refractory mesial temporal lobe epilepsy(mTLE). Li et al. and Gong 
et al. reported downregulation of miRNA-153 in brain tissue and plasma of mTLE patients.^{8, 9} Also, Jiang et al. reported that expression of miRNA-199a was significantly reduced in brain tissues of intractable TLE patients. ¹⁰

Conversely, Antônio et al. found that miRNA-199a was upregulated in the blood samples from patients with mTLE with hippocampal sclerosis group when compared to that of healthy individuals. ¹⁵ This difference might be explained by variances in the study design and type of epilepsy.

The mammalian target of rapamycin (mTOR) signaling pathway was suggested to have a pivotal role in many cellular functions that may affect neuronal excitability and mediate epileptogenic tendency. mTOR is a conserved serine/threonine kinase and a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, found in two multiprotein complexes, namely mTORC1 and mTORC2. The upregulation of mTOR signaling is a common incident in epilepsy. ¹⁶ miRNA-199a inhibits mTOR signaling by binding directly to the 3′UTR of mTOR. Multiple studies demonstrated that downregulation of miRNA-199a develops several malignancies and is associated with the increased activity of mTOR signaling pathway.^17–19 Also, miRNA-153 downregulation was correlated with Rictor (mRNA and protein) upregulation. Rictor is a key component of mTORC2 and is required for mTORC2 activation. ²⁰ This may explain our findings of downregulation of both miRNAs-153 and -199a in generalized epilepsy, but further studies are needed to confirm this relationship.

This study involves some limitations. First, it is doubtful if other factors may alter miRNA expression in serum, which were not considered in our research and may in turn alter the serum miRNA levels in drug-resistant epilepsy. AEDs taken by each patient are given special concern. Second, we have a limited number of patients, and further studies are still required involving a larger sample.

Conclusion

In conclusion, serum expression levels of miRNAs-153 and -199a may have a diagnostic role for generalized epilepsy. Also, decreased circulating levels of miRNA-153 and -199a are associated with a higher risk for drug-resistant epilepsy and, thus, could constitute novel noninvasive molecular markers for early diagnosis of refractory epilepsy.

Authors’ Contribution

MZ, EK, and HED were responsible for conceptualization and design of the research. MZ, HA, and HED were responsible for data collection, data analysis and interpretation. MZ and HN were responsible for laboratory techniques. All authors contributed equally in literature research, manuscript preparation, editing and review.

Statement of Ethics

Informed consent to participate in this study was obtained from each subject. The study was approved by the Local Ethics Committee of the Faculty of Medicine, University of Alexandria.

This article complies with the International Committee of Medical Journal Editors’ uniform requirements for the manuscript.