Frozen tissue samples from brain donors diagnosed with TLE (n = 15, age 11.2 $\pm$ 2.6 years, onset age 7.4 $\pm$ 1.3 years) and control individuals (n = 15, age 10.6 $\pm$ 3.7 years) were analyzed. Samples were stored at -80$^\circ$C. Three slices (400 $\mu$m thick) of hippocampus Area CA3 from TLE subjects or normal control subjects were collected by surgical excision of the epileptic focus using a manual tissue chopper. All samples were supplied by the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University. The study was approved by the Ethics Committee of the Capital Medical University (No.KY2015-006-01). Written consents were collected from all subjects before the study.

Wistar rats were purchased from the animal center of Hubei College of Traditional Chinese Medicine and housed under standard laboratory condition. One male rat was housed with a female rat. Primary hippocampal cells from 100 P0 newborn rats were cultured as described previously (Nunez:2008). Briefly, three slices of hippocampus were obtained from the brain and dissociated to single cells after digestion with 0.5% Trypsin at 37$^\circ$C for 15 minutes. Cells were then washed twice with Hank’s buffered saline solution and treated with DNase I. Neurons were placed on glass coverslips with plating medium in a humidified 37$^\circ$C incubator (5% CO₂). After confirming cell viability and that they had attached, cells were cultured with Neurobasal medium. One-third of the medium was replaced with fresh Neurobasal medium once a week.

To mimic TLE condition in children, primary hippocampal neurons from newborn rats were cultured in magnesium-free medium (145 g NaCl, 2.5 g KCl, 10 g HEPES, 2 g CaCl₂, 10 g glucose, 0.002 g glycine dissolved in 1000 mL distilled water, pH 7.2) at 37$^\circ$C for three hours, after which hippocampal epileptiform activity was induced (Lewis:1990, Sombati:1995).

miRNA and mRNA were isolated from human brain tissue and cultured rat hippocampal neurons, respectively, by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and respectively reverse transcribed by either the qScript$^{TM}$ microRNA cDNA Synthesis Kit for miRNA (Quanta BioSciences, Gaithersburg, MD, USA) or the QuantiTect Reverse Transcription Kit for mRNA (Qiagen, Valencia, CA, USA). Real-time PCR was performed using the Maxima SYBR Green/ROX qPCR Master Mix assay (Thermo Fisher Scientific, Rockford, IL, USA) with GAPDH or U6 as internal controls in the StepOnePlus system (Applied Biosystem, Foster City, CA, USA). Following amplification in triplicate for each sample, Ct values were acquired with manual thresholds by 7500 System SDS software (Applied Biosystem). Relative expression levels were normalized to the expression level of GAPDH or U6 according to the $\Delta\Delta$CT method. The expression levels between different groups were compared using 2$^{-\Delta\Delta Ct}$, while p-values $\leq$ 0.05 were considered statistically significant. Primer sequences were: miR-135a-5p: 5’-TCACATAGGAATAAAAAGCCATA-3’; Caap1 F: 5’-CTGTTCCTCCACCAGAAGATG-3’; Caap1 R: 5’-ACCAGCTTTCATTAGGGCTTTA-3’.

Western blotting was performed as previously described (Zhong:2014). Human brain tissues and cultured rat primary hippocampal neurons were sonicated in ice-cold lysis buffer according to the manufacturer’s instruction (Cell Signaling Technology, Danvers, MA, USA) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) to extract protein. Equal amounts of protein (30 $\mu$g) from each group or the Precision Plus Protein Standard used for marker (Bio-Rad Laboratories, Hercules, CA, USA) were resolved by SDS-PAGE, transferred onto PVDF membranes and immunoblotted with primary antibodies for CAAP1 (human)/Caap1 (rat) and GAPDH at 1:1000 dilutions in 5% nonfat milk. CAAP1 antibody (NBP1-86644) was obtained from Novus Biologicals (Littleton, CO, USA). HRP-conjugated goat anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) secondary antibody was used to detect primary antibody. The intensities of target protein bands were assessed by densitometry and normalized to those of GAPDH (Abcam). The signals were detected by the SuperSignal West Femto Maximum Sensitivity Substrate kits (Thermo Fisher Scientific) and the densitometric intensities were determined using Image J.

miR-135a-5p inhibitor was synthesized by Life Technologies (Grand Island, NY, USA). Cultured rat primary hippocampal neurons were transfected at a confluency of 85-90% on a 96 well plate. Prior to transfection, regular medium was replaced with serum-free Opti-MEM$^{\circledR}$ I medium. 5 $\mu$L of 100 $\mu$M miR-135a-5p inhibitor and 5 $\mu$L Lipofectamine$^{TM}$ RNAiMAX (Invitrogen, Carlsbad, CA, USA) were added into 50 $\mu$L Opti-MEM$^{\circledR}$ I medium and incubated for five minutes at room temperature. Cells were then transfected with miR-135a-5p inhibitor mixed with Lipofectamine$^{TM} $RNAiMAX and incubated at 37$^\circ$C for six hours. After transfection, Opti-MEM$^{\circledR}$ I medium was replaced with regular medium. After 24 hours cells were cultured in a magnesium-free medium at 37$^\circ$C for three hours. The sequence of miR-135a-5p inhibitor was: 5’-UCACAUAGGAAUAAAAAGCCAUA-3’.

1 $\times$ 10$^{6}$ cultured rat primary hippocampal neurons were harvested from each group and washed by incubation buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 5 mmol/L $CaCl_2$). Hippocampal neurons were then suspended in 100 $\mu$L PBS with 1 $\mu$g Annexin V-FITC (Becton Dickinson, Franklin Lakes, NJ, USA) and Propidium iodide (PI) (Thermo Fisher Scientific), followed by dark incubation at room temperature for 10-15 minutes. After a single wash with incubation buffer, SA-FLOUS was added to each sample and dark incubated at 4$^\circ$C for 20 minutes before analysis by flow cytometry.

A MTT assay kit (Abcam, Cambridge, MA, USA) was used to detect the survival of cultured rat primary hippocampal neurons (Chen:2012). Medium was discarded from the cell cultures in the 96 well plate. Then 50 $\mu$L of serum-free media and 50 $\mu$L of MTT solution were added into each well and plates were incubated at 37$^\circ$C for three hours. After incubation, 150 $\mu$L of MTT solvent was added into each well. The plate was shaken on an orbital shaker for 15 minutes and absorbance was measured at OD = 590 nm within one hour after shaking.

Data is reported as mean $\pm$ standard error ($\pm$SE). Statistical differences were determined by use of the Student’s t-test between two groups and two-way ANOVA between multiple groups. After the two-way ANOVA analysis, a Tukey’s multiple-comparison test was conducted to estimate the significance of the differences. All statistical analyses were conducted by the SigmaStat 3.5 software. A p-value < 0.05 was considered statistically significant.

The clinical characteristics of brain donors are summarized in Table. 1. Compared to the control group (n = 15), miR-135a-5p expression was significantly increased in the hippocampus of 80% of the children diagnosed with TLE (n = 15) detected by qPCR (Fig. 1A). In contrast, as the predicted target gene of miR-135a-5p (Fig. 1B), CAAP1 gene expression in hippocampal neurons from 87% of the children with TLE (n = 15) was decreased compared to that in control group (Fig. 1C and Fig. 1D). These data suggested that miR-135a-5p was also upregulated in children with TLE, as previously reported for adult cases (Alsharafi:2015). Moreover, the upregulation of miR-135a-5p suppressed the expression of its target gene CAAP1 in TLE. However, the expression of miR-135a-5p and CAAP1 were not significantly changed in all children with TLE, suggesting that other genes may be involved in TLE formation.

Figure 1.

miR-135a-5p expression is increased in temporal lobe epilepsy. A: Quantitative qRT-PCR analyses of miR-135a-5p levels in hippocampus of children. B: Prediction of miR-135a-5p binding site in Caap1 miRNA 3’-UTR by microRNA website (www.microrna.org/microrna/home.do). C: mRNA level of Caap1 in hippocampus of children. D: Protein abundance of Caap1 in hippocampus of children. qRT-PCR was performed using 15 samples from control or TLE children respectively. Experiments repeated three times (n = 3). **p < 0.05 vs. Control group.

To mimic the TLE condition in children and to further investigate the expression of miR-135a-5p and Caap1 in vitro, primary hippocampal cells from newborn rats were cultured. After three hours of treatment in magnesium-free medium, the expression of miR-135a-5p was increased significantly compared to that of control hippocampal cells (Fig. 2A). Moreover, the overexpression of miR-135a-5p repressed the expression of Caap1 in hippocampal neurons cultured in magnesium-free medium (Fig. 2B and Fig. 2C). These data suggested that miR-135a-5p expression was upregulated, whereas, Caap1 expression was decreased both in vivo and in vitro under the TLE condition.

Figure 2.

miR-135a-5p is upregulated in TLE model in newborn rat hippocampal neurons. A: Quantitative qRT-PCR analyses of miR-135a-5p levels in primary hippocampal neurons from new born rats. B: mRNA level of Caap1 in hippocampus neurons from newborn rats. C: Protein abundance of Caap1 in primary hippocampal neurons from newborn rats. Experiments repeated three times (n = 3). No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs Control group.

To determine whether miR-135a-5p mediates the pro-apoptotic effect of hippocampal cells in children with TLE, the miR-135a-5p inhibitor was employed. In cultured hippocampal neurons apoptosis was detected by flow cytometry. Treatment of magnesium-free medium dramatically triggered cell apoptosis compared to that in the control group (Fig. 3A and Fig. 3B). However, transfection of miR-135a-5p inhibitor into cells cultured in magnesium-free medium resulted in a significantly lower number of apoptotic cells (Fig. 3A and Fig. 3B). Additionally, treatment by miR-135a-5p inhibitor did not affect apoptosis of control cells (Fig. 3A and Fig. 3B). Moreover, the addition of miR-135a-5p inhibitor to cells treated with magnesium-free medium prevented the expression of Caap1 inhibited by the overexpression of miR-135a-5p (Fig. 4A and Fig. 4B). These data collectively suggest that miR-135a-5p mediates TLE-induced apoptosis of hippocampal cells through suppression of Caap1 expression.

Figure 3.

miR-135a-5p mediates the pro-apoptotic effect of TLE. A and B: FACS analysis of primary rat hippocampal cells for apoptosis and quantification of apoptotic cells per group. Cells treated with miR-135a-5p inhibitor for 24 hours before being cultured in magnesium-free medium. Annexin V labeled with FITC and PI were used to stain cells. No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs. Control group; $^{\#\#}$p < 0.05 vs. Control+miR-135a-5p inhibitor group; $^{\Delta\Delta}$p < 0.05 vs No Mg$^{2+}$ group.

Figure 4.

miR-135a-5p regulates Caap1 expression negatively in TLE model. A: mRNA level of Caap1 in primary rat hippocampus neurons from newborn rats treated with or without miR-135a-5p inhibitor. B: Protein abundance of Caap1 in hippocampus neurons from newborn rats. Experiments repeated three times (n = 3). No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs. Control group; $^{\#\#}$p < 0.05 vs Control+miR-135a-5p inhibitor group; $^{\Delta\Delta}$p < 0.05 vs. No Mg$^{2+}$ group.

To further explore the role of miR-135a-5p in children with TLE, a MTT assay was employed to detect the proliferation of hippocampal neurons. After culture in magnesium-free medium, cell survival assessed by cell metabolic activity was reduced compared to that of control cells. Conversely, transfection by the miR-135a-5p inhibitor significantly increased cell survival (Fig. 5). Moreover, the addition of miR-135a-5p inhibitor did not promote cell survival under normal condition (Fig. 5). The above data suggests that miR-135a-5p was also involved in the regulation of neuron survival in TLE.

Figure 5.

Inhibition of miR-135a-5p promotes TLE-reduced cell survival. Quantification of cell survival indicated by MTT assay in primary rat hippocampal cells. Cells treated with miR-135a-5p inhibitor for 24 hours before being cultured in magnesium-free medium. Experiments repeated three times (n = 3). No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs. Control group; $^{\#\#}$p < 0.05 vs. Control+miR-135a-5p inhibitor group; $^{\Delta\Delta}$p < 0.05 vs. No Mg$^{2+}$ group.