Dataset Information

MiR224-5p Inhibitor Restrains Neuronal Apoptosis by Targeting NR4A1 in the Oxygen-Glucose Deprivation (OGD) Model.

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.

SUBMITTER: Liu LL

PROVIDER: S-EPMC7330102 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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MiR224-5p Inhibitor Restrains Neuronal Apoptosis by Targeting NR4A1 in the Oxygen-Glucose Deprivation (OGD) Model.

Liu Ling-Ling LL Qiao Shan S Wang Mei-Ling ML Wu Huai-Kuan HK Su Yong-Xin YX Wang Ke-Mo KM Liu Xue-Wu XW

Frontiers in neuroscience 20200625

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 ...[more]

PMID: 32670010

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Project description:Neuronal apoptosis is a major pathological hallmark of the neonatal hypoxic-ischemic brain damage (HIBD); however, the role of miR-7a-2-3p in the regulation of HIBD remains unknown. The purpose of this study was to explore the possible roles of miR-7a-2-3p in brain injury using a hypoxia-ischemia model in rats and oxygen-glucose deprivation (OGD) model in vitro. Firstly, we established the hypoxia-ischemia (HI) model and verified the model using Zea Longa scores and MRI in rats. Next, the changes of miR-7a-2-3p were screened in the ischemic cortex of neonatal rats by qRT-PCR at 12, 48, and 96 h after HIBD. We have found that the expression of miR-7a-2-3p in the HI rats decreased significantly, compared with the sham group (P < 0.01). Then, we established the OGD model in PC12 cells, SH-SY5Y cells and primary cortical neurons in vitro and qRT-PCR was used to confirm the changes of miR-7a-2-3p in these cells after the OGD. In order to determine the function of miR-7a-2-3p, PC12 cells, SH-SY5Y cells and rat primary cortical neurons were randomly divided into normal, OGD, mimic negative control (mimic-NC) and miR-7a-2-3p groups. Then, Tuj1+ (neuronal marker) staining, TUNEL assay (to detect apoptotic cells) and MTT assay (to investigate cell viability) were performed. We have found that the number of PC12 cells, SH-SY5Y cells and cortical neurons in the miR-7a-2-3p groups increased significantly (P < 0.01) in comparison to the OGD groups. The survival of cortical neurons in the miR-7a-2-3p group was improved markedly (P < 0.01), while the apoptosis of neurons in the miR-7a-2-3p group was significantly decreased (P < 0.01), compared with the normal group. Lastly, we investigated the target genes of miR-7a-2-3p by using the prediction databases (miRDB, TargetScan, miRWalk, and miRmap) and verified the target genes with qRT-PCR in the HI rats. Bioinformatics prediction showed that Vimentin (VIM), pleiomorphic adenoma gene 1(PLAG1), dual specificity phosphatase 10 (DUSP10), NAD(P)H dehydrogenase, quinone 1 (NQO1) and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) might be the targets of miR-7a-2-3p and the qRT-PCR confirmed that VIM increased in the HI rats (P < 0.01). In conclusion, miR-7a-2-3p plays a crucial role in the hypoxic-ischemic injury, and is associated with regulation of VIM.

Project description:As a classical growth promoter and metabolic regulator, growth hormone (GH) is involved in development of the central nervous system (CNS). This hormone might also act as a neurotrophin, since GH is able to induce neuroprotection, neurite growth, and synaptogenesis during the repair process that occurs in response to neural injury. After an ischemic insult, the neural tissue activates endogenous neuroprotective mechanisms regulated by local neurotrophins that promote tissue recovery. In this work, we investigated the neuroprotective effects of GH in cultured hippocampal neurons exposed to hypoxia-ischemia injury and further reoxygenation. Hippocampal cell cultures obtained from chick embryos were incubated under oxygen-glucose deprivation (OGD, <5% O2, 1 g/L glucose) conditions for 24 h and simultaneously treated with GH. Then, cells were either collected for analysis or submitted to reoxygenation and normal glucose incubation conditions (OGD/R) for another 24 h, in the presence of GH. Results showed that OGD injury significantly reduced cell survival, the number of cells, dendritic length, and number of neurites, whereas OGD/R stage restored most of those adverse effects. Also, OGD/R increased the mRNA expression of several synaptogenic markers (i.e., NRXN1, NRXN3, NLG1, and GAP43), as well as the growth hormone receptor (GHR). The expression of BDNF, IGF-1, and BMP4 mRNAs was augmented in response to OGD injury, and exposure to OGD/R returned it to normoxic control levels, while the expression of NT-3 increased in both conditions. The addition of GH (10 nM) to hippocampal cultures during OGD reduced apoptosis and induced a significant increase in cell survival, number of cells, and doublecortin immunoreactivity (DCX-IR), above that observed in the OGD/R stage. GH treatment also protected dendrites and neurites during OGD, inducing plastic changes reflected in an increase and complexity of their outgrowths during OGD/R. Furthermore, GH increased the expression of NRXN1, NRXN3, NLG1, and GAP43 after OGD injury. GH also increased the BDNF expression after OGD, but reduced it after OGD/R. Conversely, BMP4 was upregulated by GH after OGD/R. Overall, these results indicate that GH protective actions in the neural tissue may be explained by a synergic combination between its own effect and that of other local neurotrophins regulated by autocrine/paracrine mechanisms, which together accelerate the recovery of tissue damaged by hypoxia-ischemia.

Project description:HECT, UBA and WWE domain-containing 1 (Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase (JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor (SP600125) or a p38MAPK inhibitor (SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38MAPK and of apoptosis-related proteins (p53, Gadd45a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018 (approval No. 2018013).

Dataset Information

MiR224-5p Inhibitor Restrains Neuronal Apoptosis by Targeting NR4A1 in the Oxygen-Glucose Deprivation (OGD) Model.

Publications

MiR224-5p Inhibitor Restrains Neuronal Apoptosis by Targeting NR4A1 in the Oxygen-Glucose Deprivation (OGD) Model.

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