Treatment with melatonin ameliorates febrile convulsion via modulating the MEG3/miR‑223/PTEN/AKT signaling pathway
- Authors:
- Published online on: June 23, 2021 https://doi.org/10.3892/ijmm.2021.4987
- Article Number: 154
Abstract
Introduction
As a type of seizure often diagnosed in childhood or infancy, febrile seizure (FS) is one of the most commonly encountered acute neurologic conditions in children (1). In the 1990s, the International League Against Epilepsy defined FS as a seizure observed in children of >1 month that is not induced by infection to the central nervous system (CNS) and does not meet the standard for other acute seizures (2).
A separate study carried out by Molina-Carballo et al (3) in 54 children with febrile and epileptic convulsive crisis showed that serum melatonin (MT) expression was enhanced during seizure attacks and returned to normal values within 1 h. The authors further concluded that excitation of MT generation by a convulsive crisis may help to achieve homeostasis in the body (3). Similar results were observed in other studies (4).
Long non-coding RNAs (lncRNAs) are long RNA transcripts that do not encode proteins (5). The expression of lncRNAs is usually specific to cell and tissue types (6,7). Thus far, lncRNAs have been reported to participate in the pathogenesis of numerous malignancies by regulating gene modification and transcription (8,9). Previous results showed that lncRNA MEG3 is enriched in the endothelium and acts as a competing endogenous RNA of microRNA (miRNA/miR)-223, while MEG3 was also suspected to participate in MT-induced prevention against atherosclerosis (10). Moreover, MEG3 can inhibit the activity of miR-223, while increasing the expression of cryopyrin (NLRP3) and the pyroptosis of endothelial cells (10).
It has been demonstrated that miR-223 harvested from macrophages can enhance the chemoresistance of EOC cells via the phosphatase and tensin homolog (PTEN)-PI3K/protein kinase B (AKT) signaling (11). PTEN was illustrated to be inhibited by miR-223 to enhance the level of PI3K/AKT activation, indicating that AKT is a key mediator in the miR-223/PTEN signaling. Additionally, miR-223 is negatively correlated with PTEN expression in various types of tumor cells, indicating the important role of PTEN in tumorigenesis (11).
PTEN has the properties of lipid phosphatase. Moreover, the substrate of PTEN is PIP3, which is hydrolyzed by PTEN to generate phosphatidylinositol 4,5-bisphosphate (PIP2) (12-15). PTEN inhibits PI3K/AKT signaling by dephosphorylating PIP3 (14). On the other hand, the inhibition of PTEN activates PI3K/AKT signaling and promotes cell survival (15). PTEN was reported to impair AKT activation by suppressing its membrane recruitment, thus suppressing the proliferation, growth and survival of cells. Therefore, PTEN plays an important role in suppressing the malignant transformation of normal cells (16).
It is commonly known that AKT is important in the regulation of numerous cellular processes, including protein translation, gene transcription, cell survival and cell proliferation (17). For example, accumulating evidence has implied that the inhibition of the kinase activity of AKT promotes FS-mediated neuronal apoptosis in the hippocampus, while FS and febrile convulsion (FC) did not affect the level of total AKT in rats, indicating that AKT/MTOR signaling is involved in FS-induced neuronal injury, and the initialization of endoplasmic reticulum stress promoted hippocampal cell apoptosis by affecting the phosphorylation of AKT (18). Furthermore, γ-aminobutyric acid (GABA), which acts as the main inhibitory signal in the peripheral nervous system and CNS, has been associated with severe neurological and neuromuscular disorders (19). There is increasing evidence suggesting that the low expression of GABA is an important trigger of seizures (20). For instance, Mackenzie and Maguire (21) reported that chronic stress-induced GABA downregulation in the mouse hippocampus can increase the susceptibility to seizure.
It has been reported that treatment with MT can downregulate the expression of lncRNA-MEG3 and thereby upregulate the expression of miR-223, a competing endogenous RNA of MEG3 (10). Furthermore, PTEN has been identified as a direct target of miR-223, while the PTEN/AKT signaling pathway has been shown to be involved in the pathogenesis of FC (18,22). In the present study, the aim was to evaluate the therapeutic effect of MT on FC and the according underlying molecular mechanisms. Accordingly, it was demonstrated that MT alleviated FC via the MEG3/miR-223/PTEN/AKT pathway, which further indicated MT as a potential novel treatment strategy for FC.
Materials and methods
Animals
In the present study, a total of 24 Wistar rats (age, 5-10 days; weight, 6-8 g) were used to mimic newborn infants delivered after full-term pregnancy in humans. In addition, brain tissues isolated from rats aged 15 to 20 days were used to mimic the brain of human infants with an age of ≤1 years old. Specifically, male Wistar rats aged 17 days were utilized to establish the animal model. During the experiments, these male Wistar rats were placed under anesthesia using a mixture of 3 mg/kg xylazine and 80 mg/kg ketamine administrated intraperitoneally. After the anesthesia of rats was successfully established, the head of each rat was opened via a small incision to remove the brain tissue. In the next step, a pair of electrodes made of stainless steel were inserted into the frontal region of the dura mater of each rat. In addition, a third electrode made of stainless steel was inserted into the occipital region of the dura mater of each rat. Subsequently, the electrodes were fixed to the skull of each rat using dental acrylic. After the operation, all rats were placed in an SPF animal facility with temperature control (the room temperature was set to 23±1°C). In addition, the light/dark cycle was set to 12/12 h, and all rats had free access to a standard chow diet and drinking water throughout the entire experiment. By the end of the experiment, the animals were killed by a lethal intravenous dose of sodium pentobarbital (100 mg/kg). Death was confirmed by various combined factors, including lack of pulse, breathing, corneal reflex, and response to toe pinch; inability to hear respiratory sounds and heartbeat. No secondary procedure was used. Moreover, percutaneous cardiac puncture was performed after the rats were unconscious, and failure of movement of the needle and attached syringe after insertion into the heart (aspiration of blood provides evidence of correct location) also indicates lack of cardiac muscle movement and death. All animal procedures were approved by Wuhan Children's Hospital (approval no. WHET20170731V1; Wuhan, China) and were in strict compliance with the 'Guide for the Care and Use of Laboratory Animals' published by the US National Institutes of Health (23).
Establishment of an animal model of FC
An animal model of FC was established. During the animal model establishment, all rats were exposed to hyperthermia by being placed in a water tank holding water with a water level (5 cm depth of water at 45°C). In addition, the rats were also exposed to hyperthermia by keeping the water tank temperature at 45°C for ~5 min until the symptoms of seizure were observed. A total of 10 such experimental operations were repeated every 2 days. For the rats of the NC group, they were placed in water of 37°C during similar experimental operations. In addition, after the rats were exposed to hyperthermia, their core temperature was immediately measured using a temperature probe via the rectum. Furthermore, the duration of seizure in each rat was determined based on the time interval when the rat was put into the water tank until the initial signs of seizure were first observed. For comparison, 10-time intervals of seizure (also named seizure latencies) were recorded in each rat. The duration of seizure in each rat was calculated as the time interval between the onset of initial seizure signs to the moment when the rat first gained consciousness, which was evaluated based on the responsiveness of each rat to a range of multiple stimuli, including cage tapping, loud clapping sound, touch and trace of object movement in front of the eyes of the rat. By the end of each experimental operation, all rats were dried with towels first and further dried underneath a heating lamp before they were placed back in their cages. In this study, the seizure intensity in each rat was evaluated. In brief, the scoring of seizure intensity was as follows: i) 0 points indicated no sign of convulsion; ii) 1 point indicated facial clonus; iii) 2 points indicated head nodding; iv) 3 points indicated forelimb clonus; v) 4 points indicated rearing; and vi) 5 points indicated rearing and back falling. In this study, animals were divided into three groups with 8 rats in each group: i) NC group (sham-operated rats treated with PBS); ii) FC group (rats suffering from FC induced by a high temperature); and iii) FC + MT group (rats were given 80 mg/kg MT via intraperitoneal injection at 15 min before the induction of seizure).
RNA isolation and reverse transcription-quantitative (RT-q) PCR
Total RNA was isolated from tissue and cell specimens using a miRNeasy Mini kit (Qiagen GmbH) according to the manufacturer's protocols. RNA concentration and purity were confirmed using an UV spectrophotometer (Eppendorf). After purified RNA was made into cDNA templates via RT reactions using a TaqMan™ RNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a 9700 PCR Machine (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the conditions described as follows: 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then kept at 4°C. The cDNA templates were then subjected to qPCR carried out on a 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a TaqMan Universal PCR Master Mix II (Applied Biosystems; Thermo Fisher Scientific, Inc.) per the kit instructions. The thermocycling conditions were as follows: 10 min at 95°C; 40 cycles for 15 sec at 95°C; and finally 60 sec at 60°C. The relative expression of MEG3, miR-223 and PTEN mRNA was calculated using the 2-ΔΔCq method (24). The primer sequences were as follows: MEG3 forward, 5′-GCCAAGCTTCTTGAAAGGCC-3′ and reverse, 5′-TTCCACGGAGTAGAGCGAGTC-3′; miR-223 forward, 5′-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGGGGT-3′ and reverse, 5′-TGTCAGTTTGTCAAATACCCCA-3′; and PTEN forward, 5′-CTGGGTACCATGACAGCCATCATCAAAG-3′ and reverse, 5′-GGCCTCGAGTCAGACTTTTGTAATTTGTG-3′. U6 (forward, 5′-GTGCTCGCTTCGGCAGCA-3′ and reverse, 5′-CAAAATATGGAACGCTTC-3′) was used as an internal reference gene for miR-223 and GAPdH (forward, 5′-CGACTTCAACAGCGACACTCAC-3′ and reverse, 5′-CCCTGTTGCTGTAGCCAAATTC-3′) was used as an internal reference gene for MEG3 and PTEN.
Cell culture and transfection
SH-SY5Y (cat. no. ATCC® CRL-2266™; American Type Culture Collection) and U251 MG cells (cat. no. 09063001; Sigma-Aldrich; Merck KGaA) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Sigma-Aldrich; Merck KGaA). The culture was carried out at 37°C and 5% CO2. To determine the effect of miR-233, SH-SY5Y and U251 cells were seeded into 24-well plates at a density of 5×105 cells/well and transfected with miR-233 mimics (5′-CCGAGGTATTCGCCATGGATACGACTGGGGT-3′; Thermo Fisher Scientific, Inc.) and scramble miRNA (5′-UGGGCGUAUAGACGUGUUACAC-3′; Thermo Fisher Scientific, Inc.), negative control (NC) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) per the recommendation of the manufacturer. SH-SY5Y cells were in the following three groups: i) NC group, cells were treated with PBS at 37°C for 24 h; ii) H2O2 group, cells were treated with H2O2 to model apoptosis at 37°C for 24 h; and iii) H2O2 + MT group, cells were treated with 1 µM MT at 37°C for 24 h.
Vector construction, mutagenesis and luciferase assay
The 3′untranslated region (UTR) of PTEN containing the miR-233 binding site was inserted into a pcDNA3.1 vector (Promega Corporation) to generate the wild-type (WT) PTEN 3′UTR vector. Then, the miR-233 binding site in the 3′UTR of PTEN was subjected to site-directed mutagenesis to generate the mutant (MUT) PTEN 3′UTR vector using a site-directed mutagenesis kit (Agilent Technologies, Inc.). Similarly, the WT and MUT vectors of MEG3 promoter containing the WT and MUT miR-233 binding sites were also generated. Subsequently, SH-SY5Y and U251 cells seeded in 24-well plates at a density of 5×105 cells/well were co-transfected with WT/MUT PTEN/MEG3 vectors in conjunction with miR-233 mimics (5′-CCGAGGTATTCGCCATGGATACGACTGGGGT-3′; Thermo Fisher Scientific, Inc.) and NC (5′-UGGGCGUAUAGACGUGUUACAC-3′; Thermo Fisher Scientific, Inc.) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), per the recommendation of the manufacturer for 48 h before the luciferase activity of transfected cells was assayed using a Bright-Glo™ Luciferase Assay System (Promega Corporation). Renilla luciferase activity was used for the normalization of the relative luciferase activity.
Western blot analysis
Protein was extracted from the SH-SY5Y and U251 MG cells using RIPA (Sigma-Aldrich; Merck KGaA) and protein concentration was determined with a BCA assay kit (Bio-Rad Laboratories, Inc.). Then, 50 µg/lane protein was resolved on a 10% NuPAGE™ Novel Bis-Tris gel (Invitrogen; Thermo Fisher Scientific, Inc.), and subsequently separated proteins were transferred to a nitrocellulose membrane (Amersham; Cytiva). The membrane was then blocked with 5% non-fat milk for 60 min at room temperature, and incubated overnight at 4°C with anti-PTEN (1:1,000; cat. no. ab267787; Abcam), anti-AKT (1:500; cat. no. ab8805; Abcam), anti-phosphorylated (p)-AKT (1:1,000; cat. no. ab38449; Abcam), anti-cleaved-caspase-3 primary antibodies (1:500; cat. no. ab32042; Abcam) and anti-β-actin (1 µg/ml; cat. no. ab8226; Abcam). β-actin was used as the loading control. Following which, membranes were further incubated for 2 h at 37°C with the appropriate secondary antibody (1:2,000; cat. no. ab6721; Abcam). After the protein bands were visualized using a SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.), the relative expression of PTEN, AKT, p-AKT and cleaved caspase-3 was calculated using Quantity One software (version 4.6.7; Bio-Rad Laboratories, Inc.).
TUNEL staining
The apoptotic status of treated samples was measured using a TUNEL staining assay kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. The samples were immersed in a 3% H2O2 solution at room temperature for 10 min, and then hydrolyzed in 20 µg/ml proteinase K solution for 20 min, washed with PBS, and treated with a citrate buffer (0.01 M) for 30 min of highpressure antigen retrieval. The samples were then processed with 50 µl TdT enzyme solution at 37°C for 1 h. Subsequently, the samples were washed with PBS and incubated with 50 µl peroxidase-labeled anti-digoxigenin at 37°C for 30 min in the dark. Neutral resin was used as the mounting medium, and a fluorescence microscope (magnification, ×400) was used to observe the samples, 10 visual fields of view were selected randomly.
Detection of seizure and duration of convulsion with ECG recordings of ß, θ, α and δ waves
Electroencephalography (EEG) signals of β, θ, α and δ waves were recorded using a NicoletOne™ Neurodiagnostic system (Natus Medical Incorporated). In brief, the EEG signal recording was evaluated in two steps: In the first step, the first 30 min of the recorded EEG signals were immediately evaluated to detect any seizure activity. In the second step, the recording was continued for ≥18 h and all recorded EEG signals were evaluated the next day. Only the seizure activity that lasted for ≥10 sec was included for the evaluation. At the end of evaluation, the entire recording of EEG signals was assessed again to carry out the trend analysis using Nicolet One software (version 32; Natus Medical Incorporated).
Statistical analysis
Statistical analysis was carried out using GraphPad Prism (v5.0; GraphPad Software, Inc.) and SPSS for Windows (16.0; SPSS, Inc.). Data is expressed as the mean ± SD. The differences among different groups were analyzed using an unpaired Student's t-test. Comparisons between multiple groups were performed via one way ANOVA, with Bonferroni's test being utilized as the post hoc test. TargetScan (http://www.targetscan.org) and PicTar-Vert (https://pictar.mdc-berlin.de) were employed to predict the potential targets of MEG3 and PTEN. Each experiment was repeated in triplicate. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression levels of MEG3, miR-223 and PTEN among the three groups
RT-qPCR was performed to detect the mRNA levels of MEG3, miR-223 and PTEN in different SH-SY5Y cell groups. As shown in Fig. 1, mRNA expression of MEG3 (Fig. 1A) was increased in the H2O2 group, while treatment with MT decreased the expression of MEG3. The expression of miR-223 (Fig. 1B) was decreased in the H2O2 group, while treatment with MT increased the expression levels of miR-223 (Fig. 1B). The mRNA expression of PTEN (Fig. 1C) showed the same tendency as MEG3.
Additionally, western blotting was used to compare PTEN, AKT, p-AKT and cleaved caspase-3 expression among the three groups. The protein level of PTEN (Fig. 1D and E) was increased in the H2O2 group, while treatment with MT decreased PTEN protein expression. The protein expression of AKT (Fig. 1D and F) was comparable among the three groups. In addition, the protein expression of p-AKT (Fig. 1D, G and H) was decreased in the H2O2 group, while treatment with MT reversed this effect. The protein level of cleaved caspase-3 (Fig. 1D and I) was increased in the H2O2 group, while treatment with MT decreased cleaved caspase-3 protein expression. The same experiment was then performed in U251 cells and similar results were observed (Fig. 2A-I).
Verification of the MEG3/miR-223/PTEN axis
TargetScan and PicTar-Vert were employed to predict the potential targets of MEG3 and PTEN. A potential binding site for miR-223 was found in MEG3 (Fig. 3A) and PTEN (Fig. 3B), indicating that MEG3 or PTEN may be direct targets of miR-223. To confirm the putative miR-223 binding site in MEG3 and PTEN, vectors containing WT or MUT MEG3/PTEN were constructed. Then, SH-SY5Y and U251 cells were co-transfected with miR-223 mimics or miR-223 NC. The successful transfection of miR-223 mimics was validated by the evidently upregulated miR-223 expression in SH-SY5Y (Fig. S1A) and U251 cells (Fig. S1B). Moreover, as shown in Fig. 3C, in SH-SY5Y cells, WT MEG3 significantly decreased the relative luciferase activity of miR-223, suggesting that miR-223 could directly bind to MEG3. Also, WT PTEN significantly decreased the relative luciferase activity of miR-223 in SH-SY5Y cells, suggesting that PTEN could directly bind to miR-223 (Fig. 3D). Additionally, the same results were observed in U251 cells (Fig. 3E and F). Taken together, these findings indicated that MEG3, miR-223 and PTEN formed a regulatory axis.
Expression levels of MEG3, miR-223 and PTEN among the three animal model groups
Animals were divided into three groups: NC, FC and the FC + MT. Then, RT-qPCR was performed to detect the mRNA expression levels of MEG3, miR-223 and PTEN. Western blotting was used to examine protein expression levels of PTEN, AKT, p-AKT and cleaved caspase-3. As shown in Fig. 4A-I, the results were consistent with those observed in cells.
To quantify neurological deficits, TUNEL staining was performed to visualize neuronal apoptosis. The results showed that the FC group had a notably higher number of apoptotic neurons than the control group. However, compared with the FC group, MT treatment could markedly decrease the number of apoptotic neurons (Fig. 5).
Detection of seizure/convulsion duration among the different animal groups
The seizure/convulsion duration in different groups on the same day were detected, as shown in Fig. 6. The duration of convulsion (except the first episode of convulsion) in the FC group was significantly higher than those in the FC + MT group.
ECG recordings were then taken for the three groups. As shown in Fig. 7A, there was no significant difference in the first b power ratios among all groups. However, the last β power ratios of FC group and FC + MT group were significantly decreased compared with the first β power ratios of these two groups. The last β power ratio of the FC group was significantly lower than the last β power ratio in the NC group, whereas treatment with MT further decreased the last β power ratio.
As shown in Fig. 7B, there was no significant difference in the first a power ratios among all groups. The last α power ratios of FC group and FC + MT group were significantly increased compared with the first α power ratios of these two groups. The last α power ratio of the FC group was significantly higher than the last α power ratio in the NC group, while treatment with MT further increased the last α power ratio. The same phenomenon was observed in the first θ power ratios, last θ power ratios (Fig. 7C), first δ power ratios and last δ power ratios (Fig. 7D).
Discussion
MT is a neuroendocrine factor produced by the pineal gland (25,26). MT is involved in a wide range of biological functions in the body, including the regulation of circadian rhythm, inflammation, sleep and immunity, as well as having roles in cancer development (27-29). Of note, it has been suggested that MT can alleviate different cardiovascular diseases, such as myocardial ischemia-reperfusion injury, atherosclerosis, hypertension and heart failure (30-32). Previous studies also reported that MT can alleviate atherosclerosis through the MEG3/miR-223/NLRP3 signaling pathway (10). In the present study, it was found that miR-223 could bind to MEG3 and was negatively regulated by MEG3. PTEN was a direct target of miR-223 and was negatively regulated by miR-223.
MEG3 is a lncRNA that carries several miRNAs. Furthermore, it has been demonstrated that miR-223 can directly bind to PTEN to induce chemoresistance in a wide range of tumor cells (33,34). PTEN has been illustrated to inhibit cell survival while promoting apoptosis (34,35). PTEN is also a negative mediator of the PI3K/AKT signaling pathway and is implicated in a wide range of malignancies (36-38). In particular, PTEN expression is dysregulated in Alzheimer's disease (AD) (39). In addition, PTEN nitrosylation is enhanced in early AD (40). Moreover, PTEN dysfunction was found to cause abnormal social behavior in mice (41).
PTEN-α, which is one type of PTEN isoform, has been demonstrated to induce the production of ATP by mitochondria (42). Furthermore, inhibition of PTEN signaling was shown to reduce the permeability of mitochondria (43). Additionally, hydrogen sulfide (H2S) was reported to improve the dysfunction in cardiac contractility by inhibiting the function of AKT2, thus alleviating the apoptosis of mitochondrial cells (44). Similarly, it was observed that H2S could reduce the expression of PTEN in mitochondria, and knockdown of PTEN expression in mice has the ability to impair learning and memory, as well as inducing spontaneous seizure (45-47). Furthermore, mice with PTEN dysfunction also show defunct social behaviors (48). Thus, mice that lack PTEN may develop significant behavioral deficits and seizure (49). The results of the present study showed that MEG3, cleaved caspase-3 and PTEN were increased in FC, whereas miR-223 and p-AKT were increased. Treatment with MT could reverse the effects of FC.
The downregulation of PTEN has been demonstrated to activate PI3K/AKT signaling and promote cell survival (15,50). Furthermore, the function of PTEN is significantly affected by PI3K/AKT signaling (51,52). Moreover, the C-terminal hydrophobic motif of AGC kinases has been demonstrated to regulate the catalytic functions of AGC kinases (53,54). In particular, the activation of AKT kinase has been shown to be mediated by several receptor tyrosine kinases (RTKs), such as the RTKs related to epidermal growth factor, insulin, platelet-derived growth factor, fibroblast growth factor and vascular endothelial growth factor (55,56). Moreover, RTKs can directly activate PI3K or indirectly activate PI3K via certain adaptor proteins, including insulin receptor substrate 1/2. A previous study evaluated AKT phosphorylation at position Ser473 and illustrated that AKT phosphorylation is reduced in the hippocampus of rats suffering from recurrent FS (18).
To conclude, taken together, the present results suggested that MT prevented FC via the MEG3/miR-223/PTEN/AKT signaling pathway. Therefore, MT treatment could be considered as a novel strategy for the treatment of FC disease.
Supplementary Data
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
GW designed the study. JH supervised the study. GW, JH, HZ, SW and ZL performed the experiments and drafted the manuscript. SH performed the experiments and analyzed the data. ZL performed the literature research, GW and JH drafted and reviewed the manuscript. GW and SW confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal procedures were approved by the Wuhan Children's Hospital (Wuhan, China) and were in strict compliance with the 'Guide for the Care and Use of Laboratory Animals' published by the US National Institutes of Health.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
Not applicable.
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