Abnormal expression and role of MicroRNA-214-3p/SLC8A1 in neonatal Hypoxic-Ischaemic encephalopathy

Abstract

Neonatal hypoxic-ischaemic encephalopathy (HIE) refers to brain damage caused by intra-uterine distress and asphyxia/hypoxia during the perinatal and neonatal periods. MicroRNA (MiR)-214-3p plays a critical role in cell growth and apoptosis. The aim of this study was to investigate the expression and role of miR-214-3p in neonatal HIE development, and to explore the underlying mechanisms. The expression of miR-214-3p was significantly down-regulated, while that of Slc8a1, a direct target of miR-214-3p, was significantly up-regulated, in the brain tissue of neonatal HIE rats. The over-expression of miR-214-3p promoted the proliferation and inhibited the apoptosis of neurones, while its down-regulation had the opposite effect. Our results indicate that miR-214-3p expression was down-regulated in neonatal HIE rats, and the up-regulation of miR-214-3p expression protected against HIE development by inhibiting neuronal apoptosis.

Keywords: MiR-214-3p; apoptosis; neonatal hypoxic-Ischaemic encephalopathy; neuronal.

FIGURE 1

Hypoxic‐Ischaemic Encephalopathy (HIE) Exacerbated Brain Injury and Neuronal Apoptosis. (A) HIE caused severe infarcts in rat brains, as shown by 2, 3, 5‐triphenyl‐tetrazolium chloride (TTC) staining. (B) Haematoxylin and eosin staining in the infarct zone after hypoxic‐ischaemic (HI) injury (original magnification, ×400). (C) Cell apoptosis in brain tissue analysed using the terminal transferase‐mediated DNA end labelling (TUNEL) assay (original magnification, ×200) (TUNEL [green]/4′, 6‐diamidino‐2‐phenylindole [DAPI] [blue]). (D–F) Western blotting detected NSE and S100B protein levels in the brain tissue of HIE rats. (G) MiR‐214‐3p levels in the brain tissue of HIE rats measured by quantitative reverse‐transcription polymerase chain reaction. Data are expressed as mean ± standard deviation. **p < .01, ***p < .001 vs. control (n = 6 rats/group).

FIGURE 2

Oxygen–Glucose Deprivation (OGD)‐Induced Neuronal Injury. An OGD model of primary neurones was developed. (A) Cell viability was determined by 3‐[4, 5‐dimethylthiazole‐2‐yl]‐2, 5‐diphenyltetrazolium bromide assay (MTT). (B,C) Cell apoptosis was analysed by flow cytometry, and the apoptosis rate was calculated. (D) Protein levels of Bcl‐2 and Caspase‐3 were detected using western blotting. (E) MiR‐214‐3p levels in the neurones were measured using quantitative reverse‐transcription polymerase chain reaction. Data are expressed as mean ± standard deviation. **p < .01, ***p < .001 vs. control (n = 6 rats/group).

FIGURE 3

Effect of MiR‐214‐3p on Oxygen–Glucose Deprivation (OGD)‐Induced Neuronal Viability. (A,B) Levels of miR‐214‐3p in the neurones of different groups were measured by quantitative reverse‐transcription polymerase chain reaction. (C,D) Cell viability was determined by 3‐[4, 5‐dimethyl‐thiazole‐2‐yl]‐2, 5‐diphenyltetrazolium bromide (MTT) assay. Data are expressed as mean ± standard deviation. **p < .01 vs. control or OGD (n = 6 rats/group). NCI, inhibitor control group; NCM, mimic control group.

FIGURE 4

Effect of MiR‐214‐3p on Oxygen–Glucose Deprivation (OGD)‐Induced Neuronal Apoptosis. (A, C) Protein levels of Bcl‐2 and Caspase‐3 in the neurones of different groups. (B, D) Messenger RNA levels of Bcl‐2 and Caspase‐3 in the neurones of different groups. (E) Cell apoptosis was analysed by flow cytometry, and the apoptosis rate was calculated. Data are expressed as mean ± standard deviation. **p < .01 vs. OGD (n = 6 rats/group). NCI, inhibitor control group; NCM, mimic control group.

FIGURE 5

Relationship Between MiR‐214‐3p and SLC8A1. (A) Interaction between miR‐214‐3p and the 3′‐untranslated region (UTR) of Slc8a1. (B) Luciferase activity of a reporter containing the wild‐type/mutant Slc8a1 3′‐UTR. ‘MUT‐SLC8A1’ indicates the Slc8a1 3′‐UTR with a mutation in the miR‐214‐3p binding site. Data are expressed as mean ± standard deviation of three independent experiments. **p < .01 vs. control (NC). (C) SLC8A1 protein levels in the brain tissue of hypoxic‐ischaemic encephalopathy (HIE) rats, as detected by western blotting. (D) SLC8A1 protein levels determined by western blotting in oxygen–glucose deprivation (OGD)‐treated neurones. (E, F) SLC8A1 protein levels determined by western blotting in OGD‐treated neurones in different groups. (G,H) Slc8a1 messenger RNA levels measured by quantitative reverse‐transcription polymerase chain reaction in OGD‐treated neurones in different groups. Data are expressed as mean ± standard deviation. **p < .01 vs. control or OGD (n = 6 rats/group). NCI, inhibitor control group; NCM, mimic control group.