Abstract
This study was designed to investigate the molecular mechanism of stroke and to explore the effect of miR-224-5p in hypoxic cortical neurons. Firstly, we established a middle cerebral artery occlusion (MCAO) model with Sprague–Dawley rats. Triphenyltetrazolium chloride (TTC) staining showed the brain infarction of an MCAO rat. Longa scores of rats were significantly increased in 12th, 24th, and 48th hours after MCAO. Then, we found that miR-224-5p was increased after MCAO in rats by qRT-PCR. In order to investigate the effect of miR-224-5p in hypoxic neurons, we established an oxygen-glucose deprivation (OGD) model with cortical neurons. MiR-224-5p was also upregulated in neurons after OGD by qRT-PCR. After transfection of the miR-224-5p inhibitor, the number of neurons in the anti-miR-224-5p group significantly increased (P < 0.01) in comparison to the anti-NC group. Furthermore, Tuj1+ (neuronal marker) staining and TUNEL assay (to detect apoptotic cells) were performed in neurons. The survival of neurons in the anti-miR-224-5p group was significantly improved (P < 0.01), while the apoptosis of neurons in the anti-miR-224-5p group was significantly decreased (P < 0.01), when compared with that of the anti-NC group. In addition, we predicted that potential target genes of miR-224-5p were nuclear receptor subfamily 4 group A member 1 (NR4A1), interleukin 1 receptor antagonist (IL1RN), and ring finger protein 38 (RNF38) with bioinformatics databases, such as TargetScan, miRDB, miRmap, and miRanda. The result of qRT-PCR confirmed that NR4A1 was significantly decreased after hypoxic injury (P < 0.01). Meanwhile, luciferase reporter’s assay indicated that NR4A1 was the direct target of miR-224-5p. Compared with the anti-miR-224-5p + siNC group, the number of cortical neurons and the length of the neuron axon in the anti-miR-224-5p + si-NR4A1 group were significantly decreased (P < 0.01), and the number of neuronal apoptosis in the anti-miR-224-5p + si-NR4A1 group was increased (P < 0.01). In conclusion, miR-224-5p played a crucial role in hypoxic neuron injury through NR4A1, which might be an important regulatory mechanism in OGD injury of neurons.
Highlights
Stroke is caused by cerebral ischemia and hypoxia, which can severely affect the health and has high mortality and morbidity (Michalski et al, 2011; Ferrell et al, 2014; Sun et al, 2017)
We found that miR-224-5p significantly increased in 12th, 24th, and 48th hours after middle cerebral artery occlusion (MCAO) in rats (n = 6, Figure 1C)
We found that the mimic of miR-224-5p could promote the expression of miR-224-5p and the inhibitor of miR-224-5p could inhibit the expression of miR-224-5p (n = 6, ∗∗P < 0.01, FIGURE 2 | Inhibition of MiR-224-5p Improved the Morphology and Increased the Number of Primary Cortical Neurons After oxygen-glucose deprivation (OGD). (A) The images of transfection of Cy3 in neurons. (B) The transfection efficiency of neurons. (C) The mimic of miR-224-5p could promote the expression of miR-224-5p, and the inhibitor of miR-224-5p could inhibit the expression of miR-224-5p (∗∗P < 0.01). (D) The images of primary cortical neurons in the control group, OGD group, mimic-NC group, anti-NC group, miR-224-5p group, and anti-miR-224-5p group. (E) Inhibition of miR-224-5p increased the number of neurons after OGD injury (∗∗P < 0.01)
Summary
Stroke is caused by cerebral ischemia and hypoxia, which can severely affect the health and has high mortality and morbidity (Michalski et al, 2011; Ferrell et al, 2014; Sun et al, 2017). As important regulators of gene expression at the post-transcriptional level (Bartel, 2004), miRNAs have complex and diverse regulatory functions in cells (Cui et al, 2006). They are involved in a variety of biological processes, such as proliferation, differentiation, metabolism, and cell apoptosis (Ambros, 2004; Bagga et al, 2005; Nohata et al, 2011). The molecular mechanism of miR-224-5p on hypoxic neuron is unclear
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