Overexpression of miR-582-5p Inhibits the Apoptosis of Neuronal Cells after Cerebral Ischemic Stroke Through Regulating PAR-1/Rho/Rho Axis

https://doi.org/10.1016/j.jstrokecerebrovasdis.2018.09.023Get rights and content

Abstract

Objective

The purpose of this study was to explore the role of miR-582-5p/proteinase-activated receptors type I (PAR-1)/Rho/Rho in neuronal cell apoptosis after cerebral ischemic stroke (CIS).

Methods

In vivo mouse model of CIS induced by middle cerebral artery occlusion and in vitro model induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in N2A cells was established. The expressions of miR-582-5p, PAR-1, RhoA, and ROCKII in brain tissues and N2A cells were detected. Neuronal cell apoptosis was detected by flow cytometry.

Results

We found that miR-582-5p expression was decreased and the expressions of PAR-1, RhoA, and ROCKII were increased in CIS mice and OGD/R model. Moreover, miR-582-5p negatively regulated PAR-1, and overexpression of miR-582-5p inhibited the activation of Rho/Rho pathway by downregulating PAR-1, thus reducing OGD/R-induced neuronal cell apoptosis.

Conclusions

Our results suggested that miR-582-5p overexpression could regulate Rho/Rho-kinase signaling pathway via targeting PAR-1, thereby governing the apoptosis of neuronal cells after CIS.

Introduction

Cerebral ischemic stroke (CIS), also known as cerebral infarction, refers to the ischemic necrosis or softening of local cerebral caused by hypoxia and ischemia, which is seriously harmful to human health.1 In recent years, the incidence and mortality of CIS is increasing and the diseased populations tend to be younger. Therefore, it is urgent to find effective treatments. Neuronal cell apoptosis is an important mechanism of neuronal injury in CIS.2 Inhibiting neuronal cell apoptosis can improve neuronal injury following CIS.3 Therefore, investigation of the molecular mechanism of neuronal cell apoptosis is conducive to explore the effective targets of preventing and treating the CIS-induced neuronal injury.

MicroRNAs (miRNAs) are a class of small noncoding RNAs, which have been confirmed to play a vital role in many biological processes, including cell migration, invasion, proliferation, and apoptosis.4 Recent studies have found that miRNAs are closely related to the occurrence and development of CIS and can serve as potential targets for the diagnosis and treatment of CIS.5, 6 However, the underlying mechanisms of some miRNAs which abnormally expressed during the course of CIS have not been understood, such as miR-582-5p. MiR-582-5p has been identified as an important regulator in multiple cancers. For example, miR-582-5p could inhibit the development of colorectal carcinoma by targeting Rab27a.7 In addition, miR-582-5p was demonstrated to decrease in traumatized cortex after traumatic brain injury, but the mechanism of miR-582-5p in neuronal cell apoptosis and neuroprotective in CIS has not been reported.

Rho/Rho-kinase signaling pathway is involved in the pathogenesis of various neurological diseases, including CIS8. Ras homolog family member A (RhoA), a member of Rho family, plays an important role in neurite outgrowth, neural network formation and nervous system apoptosis. RhoA can exert the regulation on cellular biological behavior through its key downstream effector Rho-associated kinase (ROCK)9. ROCK is a kind of serine/threonine protein kinase that includes 2 subtypes (ROCKI and ROCKII), and the expression of ROCKII is widespread in the brain, muscle, cardio tissue and placenta.10 Apoptosis is an essential cause of the neuronal damage that results from cerebral ischemia. Studies showed that when Rho/Rho-kinase was inhibited, the neurons apoptosis cerebral ischemia/reperfusion could be reduced.11, 12 Han et al13 revealed that miR-431 could inhibit neurons apoptosis in a rat model of cerebral ischemia/reperfusion injury by negatively regulating the Rho/Rho-kinase signaling pathway.

The bioinformatics analysis (mirdb.org) showed that the binding sites of miR-582-5p exist in the 3′UTR (untranslated region) of proteinase-activated receptors type I (PAR-1), which has a significant role in inducing neuronal injury.14 PAR-1, also called coagulation factor 2 receptor, is a receptor of thrombin and is overexpressed in brain tissues of CIS mice.15 PAR-1 deficiency could protect against neuronal damage after cerebral hypoxia/ischemia.16 Additionally, PAR-1 could activate Rho/Rho-kinase signaling pathway.17 The above findings prompted the inference that miR-582-5p might participate in the pathogenesis of neuronal injury following CIS through regulating PAR-1/Rho/Rho.

In this study, we detected the expression of miR-582-5p and PAR-1 in CIS mice, explored the interaction between miR-582-5p and PAR-1 in neuronal cells, and further clarified the role of miR-582-5p/PAR-1/Rho/Rho in neuronal cell apoptosis.

Section snippets

Animal

C57BL/6 mice (8-week-old, 20-25 g) were purchased from Shanghai Lab. Animal research center, and housed in individual cages with free access to food and water. The study was approved by the Institutional Animal Care and Use Committee of Tianjin Union Medical Center.

Middle Cerebral Artery Occlusion Mouse Model

The cerebral artery occlusion (MCAO) model of CIS was established as described previously.18 Briefly, mice were anesthetized using pentobarbital (40 mg/kg) by intraperitoneal injection. A blunt dissection was performed to expose the

MiR-582-5p was Decreased and PAR-1 was Increased in CIS Mice and OGD/R Model

To test which miRNA was closely related to CIS, in vivo mouse model of CIS induced by MCAO was established. Twenty-four hours later, the brain tissues of mice from sham group and I/R group were collected for thrombin activity assay, qRT-PCR, and western blot. As shown in Figure 1A, the thrombin activity in I/R group was higher than that in the sham group. The results of qRT-PCR suggested that the expression of miR-582-5p and miR-216a-5p was significantly decreased in I/R group compared with the

Discussion

The role of noncoding RNAs (ncRNAs) in human diseases, including tumors, cardiovascular disease, and metabolic diseases, has attracted increasing attention in recent years. A number of papers have demonstrated ncRNAs are closely related to systemic or local inflammatory response, cell proliferation, apoptosis, and migration that are directly involved in the occurrence and development of human diseases. miRNAs, a class of ncRNAs, have been shown to participate in the pathogenesis of CIS.13 For

Conclusions

In conclusions, our results suggested that miR-582-5p overexpression could reduce CIS-induced neuronal cell apoptosis by regulating Rho/Rho-kinase signaling pathway through targeting PAR-1. These findings have documented a potential role of miR-582-5p in the pathogenesis of neuronal injury after CIS. However, our study still had some shortcomings, such as, we did not verify the molecular mechanism in animal experiments. We will improve it in further experiments.

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    Funding: This research was supported by the Foundation of Tianjin Union Medical Center (Grant No.2016YJ040).

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