LncRNA NEAT1 affects inflammatory response by targeting miR-129-5p and regulating Notch signaling pathway in epilepsy

Abstract

It is crucial to understand the molecular mechanisms involved in epileptogenesis. This study aims to investigate the role of lncRNA NEAT1, miR-129-5p and Notch signaling pathway in epilepsy. In this research, temporal lobe tissues were collected from patients with epilepsy and healthy controls. The CTX-TNA cells were treated with IL-1β to establish as epilepsy cell model, which were then manipulated the expression level of NEAT1, miR-129-5p and Notch1 to investigate their roles in the epilepsy progression. The expression levels of RNA and protein in temporal lobe tissues and epilepsy cell model were determined by RT-qPCR, western blotting or ELISA, respectively. MTT assay was utilized to analyze the cell viability. Dual-luciferase reporter assay was used to explore the interaction relationship between lncRNA NEAT1, miR-129-5p and Notch1. Silencing NEAT1 significantly reduced the expression levels of IL-6, COX-2 and TNF-α in epilepsy cell model. The overexpression of NEAT1 suppressed the expression level of miR-129-5p. Inhibiting miR-129-5p significantly increased the expression of IL-6, COX-2, TNF-α and Notch1. Furthermore, the expression levels of IL-6, COX-2 and TNF-α were increased after overexpressing Notch1 in miR-129-5p mimics-treated cells. The expression levels of Notch1, JAG1, and HES1 were decreased after transfecting with sh-NEAT1. However, compared with sh-NEAT1 group, the expression levels of Notch1, JAG1, HES1, IL-6 and TNF-α were reversed by miR-129-5p inhibition or Notch1 overexpression. The present study verified that lncRNA NEAT1 affected inflammatory response of epilepsy by suppressing miR-129-5p and further regulating Notch signaling pathway in IL-1β-induced epilepsy cell model.Abbreviations: CNS: Central nervous system; lncRNAs: Long noncoding RNAs; NEAT1: Nuclear-enriched abundant transcript 1; miRNAs: MicroRNAs; ATCC: American Type Culture Collection; DMEM: Dulbecco's Modified Eagle Medium; FBS: Fetal bovine serum; ELISA: Enzyme-linked immunosorbent assay; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; SD: Standard deviation; ANOVA: Analysis of variance; LPS: Ligand lipopolysaccharide; GLO1: Glyoxalase I.

Keywords: Epilepsy; LncRNA NEAT1; MiR-129-5p; Notch signaling pathway.

Figure 1.

NEAT1, IL-6, IL-1β and TNF-α expression levels are increased in epilepsy patients. (a). Altered expression of lncRNA NEAT1 was measured by RT-qPCR in temporal lobe tissues of epilepsy patients and normal controls. (b). Protein level of IL-6 detected by ELISA in temporal lobe tissues of epilepsy patients and normal controls. (c). Protein level of TNF-α detected by ELISA in temporal lobe tissues of epilepsy patients and normal controls. (d). Protein level of IL-1β detected by ELISA in temporal lobe tissues of epilepsy patients and normal controls. n = 6, **p < 0.01 and ***p < 0.001.

Figure 2.

NEAT1 regulates the expression level of inflammatory factors and cell viability. (a). Altered expression levels of IL-6, COX-2 and TNF-α measured by RT-qPCR. (b). Cell viability measured by MTT assay. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3.

NEAT1 targets the expression of miR-129-5p. (a). Detection of miR-129-5p expression in model cells and normal cells by RT-qPCR. (b). Analysis of binding sites of NEAT1 and miR-129-5p using bioinformatics software. (c). Analysis of the relationship between NEAT1 and miR-129-5p in astrocytes by dual-luciferase reporter assay. (d). RT-qPCR was used to detect the expression of miR-129-5p after silencing or overexpression NEAT1 in model cells. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4.

MiR-129-5p affects the expression of inflammatory cytokines in astrocytes and cell viability. (a). Detection of inflammatory factors IL-6, COX-2 and TNF-α by RT-qPCR. (b). Cell viability measured by MTT assay. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 5.

MiR-129-5p regulates the Notch signaling pathway. (a). Detection of Notch1 expression in model cells and normal cells by RT-qPCR. (b). Analysis of binding sites of miR-129-5p and Notch1 using bioinformatics software. (c). Analysis of the relationship between miR-129-5p and Notch1 in astrocytes by dual-luciferase reporter assay. (d). The expression of Notch1 in mimic NC, miR-129-5p mimics, inhibitor NC and miR-129-5p inhibitor groups measured by RT-qPCR. **p < 0.01 and ***p < 0.001.

Figure 6.

MiR-129-5p regulates cell inflammation via the Notch pathway. (a). The expression levels of JAG1 and HES1 measured by RT-qPCR. (b). The protein expression of Notch1, JAG1, HES1 measured by Western blotting. (c). Detection of inflammatory factors IL-6, COX-2 and TNF-α by RT-qPCR in mimics NC, miR-129-5p mimics and miR-129-5p mimics +pcDNA3.1-Notch1 groups. (d). Cell viability measured by MTT assay. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 7.

NEAT1 regulates inflammatory response via miR-129-5p affecting Notch pathway. (a). The mRNA level of Notch1 measured by RT-qPCR. (b). The protein expression of Notch1, JAG1, HES1 measured by Western blotting. (c). Detection of inflammatory factors IL-6 and TNF-α by ELISA. D. Cell viability measured by MTT assay. *p < 0.05, **p < 0.01 and ***p < 0.001.