MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3.

Nan Zhang,Yun Li,Junfa Li,Song Han,Yanling Yin,Jie Zhong

doi:10.3390/ijms17091427

Abstract

miRNAs have been linked to many human diseases, including ischemic stroke, and are being pursued as clinical diagnostics and therapeutic targets. Among the aberrantly expressed miRNAs in our previous report using large-scale microarray screening, the downregulation of miR-378 in the peri-infarct region of middle cerebral artery occluded (MCAO) mice can be reversed by hypoxic preconditioning (HPC). In this study, the role of miR-378 in the ischemic injury was further explored. We found that miR-378 levels significantly decreased in N2A cells following oxygen-glucose deprivation (OGD) treatment. Overexpression of miR-378 significantly enhanced cell viability, decreased TUNEL-positive cells and the immunoreactivity of cleaved-caspase-3. Conversely, downregulation of miR-378 aggravated OGD-induced apoptosis and ischemic injury. By using bioinformatic algorithms, we discovered that miR-378 may directly bind to the predicted 3′-untranslated region (UTR) of Caspase-3 gene. The protein level of caspase-3 increased significantly upon OGD treatment, and can be downregulated by pri-miR-378 transfection. The luciferase reporter assay confirmed the binding of miR-378 to the 3′-UTR of Caspase-3 mRNA and repressed its translation. In addition, miR-378 agomir decreased cleaved-caspase-3 ratio, reduced infarct volume and neural cell death induced by MCAO. Furthermore, caspase-3 knockdown could reverse anti-miR-378 mediated neuronal injury. Taken together, our data demonstrated that miR-378 attenuated ischemic injury by negatively regulating the apoptosis executioner, caspase-3, providing a potential therapeutic target for ischemic stroke.

Highlights

Stroke represents a major cause of disability and the second leading cause of death worldwide, with an incidence of about 17 million per year
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were employed to evaluate the effect of miR-378 on oxygen-glucose deprivation (OGD)-induced cell survival and cell death, respectively. 3 h OGD/24 h reoxygenation resulted in obvious N2A cell death
As we know that TUNEL-positive cells include some necroptotic cells, we further examined the activation of caspase-3 using a cleaved-caspase-3 specific antibody

Summary

Introduction

Stroke represents a major cause of disability and the second leading cause of death worldwide, with an incidence of about 17 million per year. Extensive research has demonstrated that stroke triggers complex cellular biochemical events, and leads to neuronal cell acute necrosis, programmed necrosis (necroptosis), apoptosis, and autophagy in the ischemic brain [4,5]. It is still unclear the precise mechanisms underlying stroke-induced cell death and neurological dysfunction. To elucidate the cellular and molecular mechanisms of neural death will doubtless benefit the development of neuroprotective drugs for stroke

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