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

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

doi:10.3389/fneur.2021.719354

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

Front Neurol. 2021 Sep 10:12:719354. doi: 10.3389/fneur.2021.719354. eCollection 2021.

Authors

Chunxiao Wu^{1

2}, Lijun Zhao¹, Xinrong Li¹, Yingshan Xu³, Hongji Guo³, Zifeng Huang¹, Qizhang Wang¹, Helu Liu¹, Dongfeng Chen², Meiling Zhu¹

Affiliations

¹ Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine, Guangzhou University of Chinese Medicine, Guangdong, China.
² The Research Center of Basic Integrative Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China.
³ Clinical Medical of Acupuncture, Moxibustion and Rehabilitation, Guangzhou University of Chinese Medicine, Guangzhou, China.

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.

Keywords: bioinformatics analysis; electroacupuncture; ischemic stroke; miRNA-mRNA networks; molecular mechanisms.