Research Paper
MicroRNA-193b-3p alleviates focal cerebral ischemia and reperfusion-induced injury in rats by inhibiting 5-lipoxygenase expression

https://doi.org/10.1016/j.expneurol.2020.113223Get rights and content

Highlights

  • The miR-193b-3p was significantly up-regulated in the rat penumbra cerebral cortex responding to a ischemic stroke.

  • miR-193b-3p could bind with the 3’UTR-5-LOX-mRNA and suppress its translation.

  • Exogenous miR-193b-3p could reduce rat brain infarct size and promote neurological recovery after ischemic stroke.

  • 5-LOX is a downstream component in the miR-193b-3p neuroprotective pathway.

Abstract

Aims

Ischemic stroke has become one of the main causes of death worldwide. MicroRNAs (miRNAs) have been implicated in cerebral ischemia-reperfusion (I/R) injury and could serve as therapeutic targets. 5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes and has been implicated in inflammatory central nerve system disorders. The objective of this study was to explore the neuroprotective effects of miR-193b-3p against focal cerebral I/R injury in rats by regulating 5-LOX expression.

Methods and materials

Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion injury. The level of miR-193b-3p expression was observed in the rat cortical peri-infarct region after focal cerebral I/R injury. Bioinformatics analysis was used to predict the binding sites of miR-193b-3p, and a dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX mRNA and miR-193b-3p. Then, rats were injected with a miR-193b-3p agomir (modified and enhanced mimic) or antagomir (modified and enhanced inhibitor) in the right lateral ventricle of the brain. Neurological deficit scores, infarct volumes, neuron damage and 5-LOX enzymatic activity and expression were measured. In an in vitro experiment, cultured PC12 cells were exposed to oxygen–glucose deprivation and reperfusion (OGD/R). OGD/R-induced cells were treated with a miR-193b-3p mimic or inhibitor and 5-LOX siRNA. Cell viability, lactate dehydrogenase release, apoptosis rate and 5-LOX expression were evaluated.

Results

The level of miR-193b-3p expression was increased in the cortical peri-infarct region of rats with cerebral focal I/R injury. The results of the dual-luciferase reporter gene assay showed that a miR-193b-3p binding site was located in the 3′ untranslated region (3’UTR) of 5-LOX mRNA. Neurological deficit scores, infarct volumes and neuronal injury were alleviated by miR-193b-3p agomir treatment but aggravated by miR-193b-3p antagomir. Furthermore, leukotriene B4, cysteinyl-leukotrienes and 5-LOX expression in the cortical peri-infarct region of rats with focal cerebral I/R injury were also downregulated by miR-193b-3p agomir treatment but upregulated by miR-193b-3p antagomir. In PC12 cells, miR-193b-3p mimic significantly decreased OGD/R-induced cell death and reduced lactate dehydrogenase release and 5-LOX expression. In contrast, miR-193b-3p inhibitor exacerbated OGD/R-induced injury in PC12 cells. Additionally, the in vitro effects of miR-193b-3p inhibitor on OGD/R-induced cell injury were partially reversed by 5-LOX siRNA treatment.

Conclusion

MiR-193b-3p has a potentially neuroprotective effect on focal cerebral I/R-induced injury by inhibiting 5-LOX expression.

Introduction

Ischemic stroke is a severe disease with high morbidity and mortality and has become one of the main causes of death worldwide, leading to a heavy economic and mental burden for patients and their families(Benjamin et al., 2018; Tsai et al., 2013). Ischemic stroke is characterized by a sudden loss of blood supply to a portion of the brain, accompanied by a corresponding loss of neurological functions, such as paresis and aphasia(Ding et al., 2018). A few hours after the initial ischemic event, the ischemic brain can be perfused again by the self-activation of the fibrinolytic system or by intravenous thrombolytic therapy and intravascular thrombectomy. Reperfusion can induce further injury accompanied by oxidative stress and excessive apoptosis in the damaged brain. Focal cerebral ischemia-reperfusion (I/R) injury is a complicated pathological process that can be divided into two phases: initial injury and secondary injury. Even though the initial injury cannot be changed, secondary injury might be mitigated by effective therapy. Although many efforts have been made in past decades, unfortunately, to date, the available effective therapies to alleviate I/R injury are limited in clinical scenarios. In the pathological process of secondary injury, an excessive inflammatory reaction may play a critical role in the resulting neurological deficits, which cannot be effectively controlled by currently available therapies. However, this dilemma still suggests that therapy to control excessive neuroinflammation is of importance to relieve I/R injury(Rajkovic et al., 2018). Therefore, the complex biological mechanisms underlying brain damage after I/R should be further elucidated.

MicroRNAs (miRNAs) are short noncoding RNAs of 19–25 nucleotides that are thought to modulate the expression of at least 30% of all human genes(Reinhart et al., 2000). MiRNAs can bind to the 3′ untranslated region (3′UTR) of target messenger RNA (mRNA) to modulate its stabilization and protein translation. In particular, miRNAs are abundant in the central nervous system (CNS) and have been found to play a critical role in many important pathological processes and cerebral diseases, such as stroke(Liu et al., 2015). miRNA has been found to alleviate secondary brain damage by suppressing microglia activation in permanent focal cerebral ischemic injury in mice(Zhao et al., 2013). Additionally, some miRNAs may be used as potential biomarkers to diagnose CNS diseases, such as epilepsy(Wang et al., 2015) and Alzheimer's disease(Lugli et al., 2015).

MiRNA-193b-3p (miR-193b-3p, accession number in miRBase: MIMAT0035734) is a noncoding RNA with 19 bps that has been found to mediate inflammatory events in many tissues, such as adipose and kidney tissue. It has been found that miR-193b can regulate inflammation in adipose tissue through its effects on chemokine release from human adipocytes and macrophages(Arner et al., 2012). Additionally, the overexpression of miR-193b significantly attenuates the secretion of IL-6 in human adipocytes, which might be mediated via the NF-κB1/RELB pathway(Arner et al., 2012). The immuno-inflammatory reaction is involved in acute rejection after kidney transplantation, which is one of the main challenges in maintaining the allograft. MiR-193b might be involved in the inflammatory process of the rejection response after renal allograft transplantation(Maluf et al., 2014; Wilflingseder et al., 2013). Moreover, miR-193b has been predicted to target proinflammatory molecules, such as those involved in inflammatory cytokine production: Esr1, Il12rg, Cd247, and Ptgs2(Rao et al., 2015).

5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes (LTs) and has been implicated in inflammatory CNS disorders. We previously found that the overexpression of 5-LOX could promote the production of inflammatory cytokines and result in more severe neuronal damage after cerebral I/R injury(Liang et al., 2015). In the present study, we first observed the upregulation of miR-193b-3p in the cortical peri-infarct region of rats suffering from a focal I/R injury. Then, bioinformatics and dual-luciferase reporter gene assays were used to predict and confirm the binding site and inhibition of miR-193b-3p on 5-LOX-mRNA, respectively. Subsequently, we tried to observe whether miR-193b-3p could have a protective effect against focal cerebral I/R injury and its effect on 5-LOX expression as well as its downstream products. PC12 cells were used to further observe the effect of miR-193b-3p on neuronal injury and 5-LOX expression caused by oxygen–glucose deprivation and reoxygenation (OGD/R). Finally, an intervention combining a miR-193b-3p inhibitor and 5-LOX siRNA in vitro was used to prove that the protective effects of miR-193b-3p were dependent on inhibiting 5-LOX-mRNA.

Section snippets

Animal experiments

Adult male Sprague-Dawley rats (n = 77), weighing 250–280 g, were obtained from the Laboratory Animal Center of Chongqing Medical University (No. SCXK (Yu) 2010–0001). The rats were housed in standard conditions of 25 ± 1 °C, 50 ± 2% humidity on a 12/12-h light/dark cycle (light from 8:00–20:00) with ad libitum access to water and standard pellet food. All of the animal experimental procedures were approved by The Institutional Animal Ethics Committee of Chongqing Medical University. We

Effects of focal cerebral I/R on cerebral blood flow of rats

Regional cerebral blood flow (rCBF) was monitored using a laser Doppler flowmeter to confirm the occurrence of ischemia and reperfusion. When the filament was inserted into the internal carotid artery (ICA) through the external carotid artery (ECA) stump, rCBF decreased rapidly to 80% of the baseline within seconds. When the filament was withdrawn to allow reperfusion, rCBF started to increase to 70% of the baseline within seconds (Online Resource 3).

Expression of miR-193b-3p in the cerebral cortex of rats

A focal cerebral I/R model was successfully

Discussion

Noncoding RNAs consist of several major classes, including miRNA, lncRNA and circRNA. MiRNA is a type of noncoding RNA with less than 25 nucleotides that suppresses the expression of many mammalian genes by binding to the 3′UTR of target mRNAs(Chen and Meister, 2005). MiRNAs have been found to be involved in many diseases, such as bladder cancer(Jiang et al., 2019), asthma(Maneechotesuwan, 2019), osteosarcoma(Zhu et al., 2018) and uremia(Ketszeri et al., 2019). Moreover, miRNA has been found to

Declaration of Competing Interest

The authors declare no competing financial interests.

Acknowledgments

This work was supported by grants from Youth Foundation of the National Natural Science Foundation of China (grant No. 81801230) and the Chongqing Science and Technology Commission Fund (cstc2017shms-zdyf0142). We offer special acknowledgement to the “Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology” for providing an experimental platform.

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