HDAC9 Silencing Exerts Neuroprotection Against Ischemic Brain Injury via miR-20a-Dependent Downregulation of NeuroD1.

Liangjun Zhong,Jinxiang Yan,Lei Meng,Haitao Li

doi:10.3389/fncel.2020.544285

Abstract

Cerebral stroke is an acute cerebrovascular disease that is a leading cause of death and disability worldwide. Stroke includes ischemic stroke and hemorrhagic strokes, of which the incidence of ischemic stroke accounts for 60–70% of the total number of strokes. Existing preclinical evidence suggests that inhibitors of histone deacetylases (HDACs) are a promising therapeutic intervention for stroke. In this study, the purpose was to investigate the possible effect of HDAC9 on ischemic brain injury, with the underlying mechanism related to microRNA-20a (miR-20a)/neurogenic differentiation 1 (NeuroD1) explored. The expression of HDAC9 was first detected in the constructed middle cerebral artery occlusion (MCAO)-provoked mouse model and oxygen-glucose deprivation (OGD)-induced cell model. Next, primary neuronal apoptosis, expression of apoptosis-related factors (Bax, cleaved caspase3 and bcl-2), LDH leakage rate, as well as the release of inflammatory factors (TNF-α, IL-1β, and IL-6) were evaluated by assays of TUNEL, Western blot, and ELISA. The relationships among HDAC9, miR-20a, and NeuroD1 were validated by in silico analysis and ChIP assay. HDAC9 was highly-expressed in MCAO mice and OGD-stimulated cells. Silencing of HDAC9 inhibited neuronal apoptosis and inflammatory factor release in vitro. HDAC9 downregulated miR-20a by enriching in its promoter region, while silencing of HDCA9 promoted miR-20a expression. miR-20a targeted Neurod1 and down-regulated its expression. Silencing of HDAC9 diminished OGD-induced neuronal apoptosis and inflammatory factor release in vitro as well as ischemic brain injury in vivo by regulating the miR-20a/NeuroD1 signaling. Overall, our study revealed that HDAC9 silencing could retard ischemic brain injury through the miR-20a/Neurod1 signaling.

Highlights

Stroke is the commonest cause of death in China and the second reason for death globally, bringing high morbidity, disability, and high cost of treatment, which is often classified into ischemic stroke and hemorrhagic stroke (Pandian et al, 2018; Yao et al, 2019)
We checked the expression of histone deacetylase 9 (HDAC9) in different tissues and cells in the bioGPS website and found that HDAC9 was highly expressed in dendritic cells, macrophages, microglia, neuro2a1 cells, hippocampal neurons, and olfactory bulb, especially in the olfactory bulb and hippocampal tissues (Supplementary Figure 1) To understand the involvement of HDCA9 in ischemic brain injury, a mouse model of ischemic brain injury was established
To understand the role of HDAC9 in the oxygen-glucose deprivation (OGD)-induced cell model, we performed a series of assays in vitro and found that, OGD treatment led to an increase in cell apoptosis, Lactate Dehydrogenase (LDH) leakage rate, and the expression of inflammatory factors (TNF-α, IL-1β, IL-6; Figures 1F–H, and Supplementary Figure 2A), indicating that the OGD cell model was successfully constructed

Summary

Introduction

Stroke is the commonest cause of death in China and the second reason for death globally, bringing high morbidity, disability, and high cost of treatment, which is often classified into ischemic stroke and hemorrhagic stroke (Pandian et al, 2018; Yao et al, 2019). The HDACs family has multiple subtypes, and the roles and mechanisms of different subtypes are different (Aune et al, 2015) It is worth having an in-depth understanding of the role of each subtype in cerebral infarction, which can further reveal the pathophysiology of ischemic cerebral infarction. The expression of HDAC3/6 is increased in cerebral infarction models, and its knockout or inhibitor attenuates the neurological damage after infarction (Chen et al, 2012) The expression of another subtype of HDAC, HDAC4, is elevated in middle cerebral artery (MCA) occlusion (MCAO) and oxygen-glucose deprivation (OGD) models, and it is involved in the reconstruction of synapses and nerve repair after the injury (Kassis et al, 2015; Yuan et al, 2016). The possible involvement of histone deacetylase 9 (HDAC9) in cerebral infarction has not been thoroughly studied, recent genomic correlation studies have found that HDAC9 variant genes may be closely related to the outcome of aortic stroke (Azghandi et al, 2015; Qingxu et al, 2016), suggesting that HDAC9 may be involved in the pathophysiology of ischemic cerebral infarction

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