microRNA-182 Negatively Influences the Neuroprotective Effect of Apelin Against Neuronal Injury in Epilepsy

Abstract

Purpose: To explore the neuroprotective effects and mechanisms of Apelin (APLN), and to study the regulation of APLN expression by microRNA (miRNA) in epilepsy.

Materials and methods: In vitro and in vivo epileptic models were established with hippocampal neurons and Wistar rats. Apoptosis of neurons was identified by flow cytometry. Western blotting was used to detect the expression of proteins, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to analyze the expression of miRNA and messenger RNA (mRNA). Bioinformatics software was used to predict target genes of miRNA, which were confirmed by dual-luciferase reporter gene system and functional experiments.

Results: Our study demonstrated protective effects of APLN against neuronal death in epilepsy both in vitro and in vivo. The underlying mechanisms involved are inhibiting the expression of metabotropic glutamate receptor 1 (mGluR1), Bax, and caspase-3; promoting the expression of Bcl-2; and increasing phosphorylated-AKT (p-AKT) levels in neurons. For the first time, we found that miR-182 could negatively regulate both transcriptional and translational levels of APLN, and that the up-regulation of miR-182 inhibited the expression of APLN and Bcl-2, and promoted the expression of Bax and caspase-3.

Conclusion: APLN could protect the neurons from injury in epilepsy by regulating the expression of apoptosis-associated proteins and mGluR1 and increasing p-AKT levels, which were attenuated by miR-182. Hence, miR-182/APLN may be potential targets for epilepsy control and treatment.

Keywords: apelin; epilepsy; miR-182; neuroprotective effects; regulation.

Figure 1

Protocol used for in vitro experiments in this study.

Figure 2

Protective effect of APLN against apoptosis of neurons. Hippocampal neurons of epileptic models were transfected with pBI-CMV3-APLN overexpression, short hairpin RNA negative control (shRNA-NC), or interference APLN shRNA plasmids. Forty-eight hours post-transfection, apoptosis was determined by flow cytometry. (A) Representative figure of flow cytometry; (B) Statistical results of these groups. Data are presented as mean ± SEM of three independent experiments. ns, non-significant; ****P < 0.0001.

Figure 3

Influence of APLN on the expression of apoptosis-associated proteins, mGluR1, and p-AKT levels. Hippocampus neurons of epileptic models were transfected with pBI-CMV3-APLN overexpression, short hairpin RNA negative control (shRNA-NC), or interference APLN shRNA plasmids. Forty-eight hours post-transfection, protein expression was evaluated by Western blotting. β-actin was used as a loading control. (A) Western blotting was used to examine the protein levels of mGluR1, p-AKT, Bax, caspase-3, and Bcl-2. Statistical results of mGluR1 expression (B), p-AKT levels (C), Bax expression (D), caspase-3 expression (E), and Bcl-2 expression (F) in different groups. ns, non-significant; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Detection of luciferase activity by dual-luciferase reporter gene. Neurons were transfected with miR-182 mimics combined with control, wide-type APLN, and mutated APLN in miR-182 binding site report vectors. *P < 0.05, **P < 0.01.

Figure 5

Relative expression of miR-182 and APLN in neurons. Neurons were transfected with miRNA negative control (miR-NC), miR-182 mimics, or miR-182 inhibitors. Twenty-four hours post-transfection, the expression of miR-182 was examined by qRT-PCR, and the expression of APLN was examined by qRT-PCR and Western blotting. (A) Relative expression of miR-182 determined by qRT-PCR. (B) Relative expression of APLN gene determined by qRT-PCR. (C, D) Western blotting analysis for APLN protein expression. **P < 0.01, ****P < 0.0001.

Figure 6

Impact of miR-182 on apoptosis of neurons. Epileptic neurons were transfected with miR-NC, miR-182 inhibitor, and miR-182 mimics. Twenty-four hours post-transfection, apoptosis was determined by flow cytometry. (A) Representative figure of flow cytometry; (B) Statistical results of these three groups. Data are presented as mean ± SEM of three independent experiments. ns, non-significant; **P < 0.01.

Figure 7

Influence of miR-182 on the expression of apoptosis-associated proteins. Neurons of epilepsy were transfected with control miR-NC, miR-182 inhibitor, and miR-182 mimics. Twenty-four hours post-transfection, protein expression was evaluated by Western blotting. β-actin was used as a loading control. (A) Western blotting was used to examine the protein levels of Bax, caspase-3, and Bcl-2. Statistical results of Bax expression (B), caspase-3 expression (C), Bcl-2 expression (D) in different groups. ns, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.