The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway

Abstract

Background The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear. Methods We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus. Results We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time. Conclusions miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

The Role of Non-coding RNAs in Viral Myocarditis

“…Additionally, MiR-98 decreased cell apoptosis by targeting the FAS/FASL gene (Zhang B. Y. et al, 2016 ), and miR-21 reduced cell apoptosis by targeting programmed cell death 4 (PDCD4) (He et al, 2013 ), a well-known apoptosis gene (Gaur et al, 2011 ; Stagakis et al, 2011 ; Junker et al, 2015 ). Furthermore, another study confirmed that miR-21 reduced cell apoptosis by targeting mitogen-activated protein kinase kinase 3 (MAP2K3) (He et al, 2019 ). Interestingly, although both studies showed that miR-21 has an anti-apoptosis effect, the expression trend of miR-21 was not consistent after CVB3 infection in different studies ( Table 2 ).…”

MicroRNA Involvement in Signaling Pathways During Viral Infection

“…For instance, miR-21 could act as a protective factor during Coxsackievirus B3 (CVB3) infection, by targeting the MAP2K3/p38 MAPK pathway. In this process, miR-21 has been shown to inhibit the expression of MAP2K3, leading to a reduced phosphorylation of the p38 MAPK, thus resulting in a significant decrease in the release of viral progeny (Figure 3; He et al, 2019). Also acting upon the same pathway are miR-744, miR-24 and miR-124, which are considered potential antiviral agents for RSV virus and influenza.…”

“…In this case, the miRNAs involved are miR-24, miR-124, and miR-744. However, besides the decrease in the activation of p38 component due to a lack of phosphorylation, there has also been demonstrated a reduction in the overall levels of the same member (McCaskill et al, 2017;He et al, 2019).…”

Potential Clinical Implications of miR-1 and miR-21 in Heart Disease and Cardioprotection

“…MiR-21 was found to play a protective role also against coxsackievirus B3 (CVB3) infection-induced heart infection, likely via targeting the MAP2K3/p-38 MAPK signaling pathway. In a mouse model of CVB3 infection, miR-21 pretreatment inactivated MAP2K3/p-38 MAPK signaling and exerted cardioprotection manifested by alleviated cell apoptosis and reduced necrosis in the heart, accompanied by reduced viral titers and the remarkably prolonged survival time of animals [172]. The overall picture of the cardioprotective action of miR-21 is outlined in Figure 4.…”

“…isoflurane-induced up-regulation of miR-21 associated with ↓PDCD4 protected cardiomyocytes against H 2 O 2 injury [166] isoflurane protected mouse hearts exposed to I/R via miR-21 and Akt/NOS/mPTP [167] trimetazidine-induced ↑miR-21 accompanied by cardioprotection against I/R, ↑p-Akt and ↑Bcl-2/Bax in rats. Cardioprotection reversed by anti-miR-21 [169] miR-21 enhanced protective effect of loperamide against H/R injury in rat cardiomyocytes associated with ↓ROS and ↓apoptosis [170] Non-pharmacological miR-1 down-regulation of miR-1 by traditional Chinese medicine Tanshinone IIA led to cardioprotection via inhibition of I/R-induced p-38 MAPK in rats [173] phenolic compound paeonol exerts cardioprotection against epirubicin-induced heart injury via regulation of miR-1, PI3K/AKT/mTOR and NF-κB [176] miR-21 resveratrol-induced up-regulation of miR-21 associated with protection against I/R in rats [168] miR transfection and delivery miR-21 adenovirus miR-21 transfection decreased IS via targeting PDCD4/AP-1 [62] adenovirus miR-21 transfection improved LV remodeling & ↓apoptosis in cardiac I/R in rats [67] lentivirus miR-21 transfection induced cardioprotection against I/R in mice manifested by ↓IS, ↓fibrosis and ↓apoptosis [64] miR-21 transfection to human cardiomyocytes ↓apoptosis via JNK/p38-MAPK/caspase-3 [68] chemically synthesized exogenous miR-21 reduced IS in mice, miR-21-induced protection was abolished with miR-21 inhibitor co-treatment [165] nanoparticle delivery of miR-21 to cardiac macrophages post-MI promoted angiogenesis, reduced hypertrophy, fibrosis, and apoptosis in the remote myocardium [75] miR-21 pretreatment exerted cardioprotection against CVB3 infection via targeting MAP2K3/p38-MAPK in mice [172] Exosomal miR miR-21 depletion of exosomal miR-21 reduced protective effect of conditioned medium in H 2 O 2 -induced oxidative stress in cardiomyocytes, and in rat hearts exposed to AMI [69] ↑miR-21 in CPC-derived exosomes prevented apoptosis in H9c2 cells via ↓PDCD4 [70] Abbreviations: I/R: ischemia/reperfusion; H/R: hypoxia/reoxygenation; AMI/MI: (acute) myocardial infarction; PDCD4: programmed cell death protein 4; AP-1: activator protein 1 (downstream molecule of PDCD4); ROS: reactive oxygen species; IS: infarct size; LV: left ventricle; CPC: cardiac progenitor cell; AF: atrial fibrillation; IPC: ischemic preconditioning; IPostC: ischemic postconditioning; RIPC: remote ischemic preconditioning; RIPerC: remote ischemic preconditioning; CVB3: coxsackievirus B3.…”