Identification of MicroRNA-Potassium Channel Messenger RNA Interactions in the Brain of Rats With Post-traumatic Epilepsy

Abstract

Background: Dysregulated expression of microRNAs and potassium channels have been reported for their contributions to seizure onset. However, the microRNA-potassium channel gene interactions in traumatic brain injury-induced post-traumatic epilepsy (PTE) remain unknown. Methods: PTE was induced in male rats by intracranial injection with ferrous chloride (0.1 mol/L, 1 μl/min) at the right frontal cortex. Electroencephalography was recorded at 60 min, as well as day 1, 7, and 30, and the behavioral seizures were assessed before injection and at different time points after injection. Rats were killed on day 30 after injection. The right frontal cortex samples were collected and subjected to high throughput messenger RNA (mRNA) and microRNA sequencing. A network of differentially expressed potassium channel mRNAs and microRNAs was constructed using OryCun2.0 and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The differential mRNA and microRNA expressions were verified using quantitative real-time-PCR. The microRNA-mRNA was subject to the Pearson correlation analysis. Results: A PTE rat model was successfully established, as evidenced by behavioral seizures and epileptiform discharges on electroencephalography in PTE rats compared with sham rats. Among the 91 mRNAs and 40 microRNAs that were significantly differentially expressed in the PTE rat brain, 4 mRNAs and 10 microRNAs were associated with potassium channels. Except for potassium calcium-activated channel subfamily N member 2, the other three potassium channel mRNAs were negatively correlated with seven microRNAs. These microRNA-mRNA pairs were enriched in annotations and pathways related to neuronal ion channels and neuroinflammation. Quantitative real-time-PCR and correlation analysis verified negative correlations in miR-449a-5p-KCNH2, miR-98-5p-KCNH2, miR-98-5p-KCNK15, miR-19b-3p-KCNK15, and miR-301a-3p-KCNK15 pairs. Conclusion: We identified microRNA-potassium channel mRNA interactions associated with PTE, providing potential diagnostic markers and therapeutic targets for PTE.

Keywords: RNA sequencing; gene annotation; microRNA; post-traumatic epilepsy; potassium channel.

Figure 1

Rats with FeCl₂ injection-induced post-traumatic epilepsy (PTE) showed epileptiform discharges on electroencephalogram (EEG). A total of 12 rats were randomly divided into sham and PTE groups (n = 6/group). Rats in PTE group were injected with 10-μl FeCl₂ (0.4 mol/L, 1 μl/min) at right frontal cortex. Sham group underwent same procedures except for injection. A 1-h EEG, including first 15 min and blocks of 10 min at 5-min intervals, was recorded at 1 h before injection and at 1 h and 1, 7, and 30 days after injection. Representative EEGs are shown. (A) Normal EEG at 60 min before injection. Multiple spikes (B), two- and three-phase sharp waves (C), 10-s continuous abnormal discharges (D), 20-min explosive abnormal discharges with occasional high-amplitude spikes (E), and a sudden high-amplitude spike (F) were observed on day 30 after injection.

Figure 2

mRNA transcriptome analysis in rat frontal lobe by RNA sequencing (RNA-Seq). (A) A volcano plot of RNA-seq transcriptome data displays gene expression values of PTE rats compared with those of sham rats. Significantly differentially expressed genes (adjusted P-value <0.05) are highlighted in red (upregulated) or blue (downregulated). Non-differentially expressed genes are highlighted in green. n = 6. (B) Heat map of 12 significantly upregulated and four significantly downregulated ion channel-related genes in PTE rats compared with sham rats. (C) Heat map of four significantly upregulated potassium channel-encoding genes in PTE rats compared with sham rats. Adjusted P-value <0.05; n = 6.

Figure 3

MicroRNA transcriptome analysis in rat frontal lobe by RNA-Seq. A volcano plot of RNA-seq microRNA transcriptome data displays miRNA expression values of PTE rats compared with those of sham rats. Significantly differentially expressed microRNAs (adjusted P-value < 0.05) are highlighted in red (upregulated) or green (downregulated). Non-differentially expressed microRNAs are highlighted in blue. n = 6.

Figure 4

Prediction of microRNA–mRNA interaction. (A) Interaction network of 1,765 microRNAs with 12 ion channel-related mRNAs. (B) Interaction network of 396 microRNAs with three potassium ion channel-encoding mRNAs. Larger dots indicate mRNAs. Smaller dots indicated microRNAs. Red indicates upregulated mRNAs or microRNAs. Green indicated downregulated mRNAs or microRNAs. Blue indicates unchanged mRNAs or microRNAs.

Figure 5

Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis of differentially expressed microRNAs associated with potassium channels. (A) miRNAs were classified into three functional categories. Fifteen biological process annotations, 10 cellular component annotations, and 10 molecular function annotations were shown P < 0.05. (B) Top 10 Kyoto Encyclopedia of Genes and Genomes pathways P < 0.05.

Figure 6

Quantitative real-time PCR verification of differentially expressed potassium channel mRNAs and microRNAs. *P < 0.05, **P < 0.01 vs. sham group. (A–C) The expression of verified mRNAs (KCNH2, KCNK15, and SLC24A4) in PTE group was significantly increased compared with the control group. (D–J) The expression of verified microRNAs (miR-449a-5p,miR-98-5p,miR-19b-3p, miR301A-3p, miR-30E-5p,miR-138-5p, and miR-139-3p) in the PTE group was significantly decreased compared with the control group.

Figure 7

Correlation analysis. Pearson correlation analysis was conducted to evaluate the correlation between microRNAs and potassium channel mRNAs. (A,B) miR-449a-5p and miR-98-5p expression were significantly and negatively correlated with KCNH2 expression; (C–E) miR-98-5p, miR-19b-3p, and miR-301a-3p expression were negatively correlated with KCNK15 expression. (F) There was no significant negative correlation between miR-138-5p and KCNK15 expression. (G) miR-30e-5p expression was positively correlated with KCNK15. (H) No significant negative correlation observed between miR-139-3p and SLC24A4 expression.