Integrated Bioinformatics Analysis of Potential mRNA and miRNA Regulatory Networks in Mice With Ischemic Stroke Treated by Electroacupuncture.

Chunxiao Wu,Zifeng Huang,Lijun Zhao,Xinrong Li,Yingshan Xu,Helu Liu,Dongfeng Chen,Qizhang Wang,Hongji Guo,Meiling Zhu

doi:10.3389/fneur.2021.719354

Chunxiao Wu, Zifeng Huang + Show 8 more

Open Access

https://doi.org/10.3389/fneur.2021.719354

Copy DOI

Abstract

Background: The complicated molecular mechanisms underlying the therapeutic effect of electroacupuncture (EA) on ischemic stroke are still unclear. Recently, more evidence has revealed the essential role of the microRNA (miRNA)–mRNA networks in ischemic stroke. However, a systematic analysis of novel key genes, miRNAs, and miRNA–mRNA networks regulated by EA in ischemic stroke is still absent.Methods: We established a middle cerebral artery occlusion (MCAO) mouse model and performed EA therapy on ischemic stroke mice. Behavior tests and measurement of infarction area were applied to measure the effect of EA treatment. Then, we performed RNA sequencing to analyze differentially expressed genes (DEGs) and functional enrichment between the EA and control groups. In addition, a protein–protein interaction (PPI) network was built, and hub genes were screened by Cytoscape. Upstream miRNAs were predicted by miRTarBase. Then hub genes and predicted miRNAs were verified as key biomarkers by RT-qPCR. Finally, miRNA–mRNA networks were constructed to explore the potential mechanisms of EA in ischemic stroke.Results: Our analysis revealed that EA treatment could significantly alleviate neurological deficits in the affected limbs and reduce infarct area of the MCAO model mice. A total of 174 significant DEGs, including 53 upregulated genes and 121 downregulated genes, were identified between the EA and control groups. Functional enrichment analysis showed that these DEGs were associated with the FOXO signaling pathway, NF-kappa B signaling pathway, T-cell receptor signaling pathway, and other vital pathways. The top 10 genes with the highest degree scores were identified as hub genes based on the degree method, but only seven genes were verified as key genes according to RT-qPCR. Twelve upstream miRNAs were predicted to target the seven key genes. However, only four miRNAs were significantly upregulated and indicated favorable effects of EA treatment. Finally, comprehensive analysis of the results identified the miR-425-5p-Cdk1, mmu-miR-1186b-Prc1, mmu-miR-434-3p-Prc1, and mmu-miR-453-Prc1 miRNA–mRNA networks as key networks that are regulated by EA and linked to ischemic stroke. These networks might mainly take place in neuronal cells regulated by EA in ischemic stroke.Conclusion: In summary, our study identified key DEGs, miRNAs, and miRNA–mRNA regulatory networks that may help to facilitate the understanding of the molecular mechanism underlying the effect of EA treatment on ischemic stroke.

Highlights

Ischemic stroke is characterized by the loss of blood and oxygen in an area of the brain, resulting in damage or death of brain neural cells
These results indicate that EA intervention could significantly modify gene expression changes in the middle cerebral artery occlusion (MCAO) model and thereby regulate the pathological and behavioral changes caused by ischemic stroke
Key miRNA-mRNA networks were identified based on RNA-seq and RT-qPCR validations, and we found that miR-425-5p-Cdk[1], mmu-miR-1186b-Prc[1], mmu-miR-434-3pPrc[1], and mmu-miR-453-Prc[1] were crucial networks regulated by EA in brain neuronal cells of ischemic stroke

Summary

Introduction

Ischemic stroke is characterized by the loss of blood and oxygen in an area of the brain, resulting in damage or death of brain neural cells. Ischemic stroke is one of the leading causes of long-term disability and death worldwide (1). It is vital to find an alternative and effective method for treating ischemic stroke. Electroacupuncture (EA) is an alternative therapy that combines acupuncture and modern electrotherapy, and it is beneficial for promoting sense and motor function recovery in ischemic stroke (6, 7). The molecular mechanisms of EA in ischemic stroke, especially the regulation of key genes and miRNA networks, are still largely unknown. A systematic analysis of novel key genes, miRNAs, and miRNA–mRNA networks regulated by EA in ischemic stroke is still absent

Methods

Results

Discussion

Conclusion