MiRNA-7a-2-3p Inhibits Neuronal Apoptosis in Oxygen-Glucose Deprivation (OGD) Model.

Zi-Bin Zhang,Ting-Hua Wang,Ya-Xin Tan,Larisa Bobrovskaya,Fei-Fei Xu,Yang Xu,Liu-Lin Xiong,Jia Liu,Xin-Fu Zhou,Qiong Zhao

doi:10.3389/fnins.2019.00016

Abstract

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.

Highlights

Neonatal ischemic hypoxic brain damage (HIBD) is caused by intrauterine asphyxia and congenital diseases and is common in neonates of perinatal period (Fenichel, 1983; DelRosso et al, 2017)
In order to investigate the mechanisms of neuronal HIBD, we established the HI model in rats which was based on the classical Rice–Vannucci method (Rice et al, 1981)
The Magnetic Resonance Imaging (MRI) showed that the right brain had significant infarction area in the HI group (Figure 1B) at 1 month after HI, which suggested the successful establishing of the HI model in vivo

Summary

Introduction

Neonatal ischemic hypoxic brain damage (HIBD) is caused by intrauterine asphyxia and congenital diseases and is common in neonates of perinatal period (Fenichel, 1983; DelRosso et al, 2017). Neonatal HIBD threatens the life of neonates, and leads to series of secondary neuronal damages after perinatal period (Gieron-Korthals and Colon, 2005), which are related to remote organ dysfunctions (Klempt et al, 1992; Perlman, 2006). MicroRNAs (miRNAs) are a class of non-coding RNAs, approximately 20–22 nucleotides in length, which are involved in regulation of gene expression at the post-transcriptional level (Moss, 2002; Shi, 2003; Bartel, 2004). It is important to explore the molecular effects of miR-7a-2-3p in HIBD

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