Wu Chunxiao, Zhao Lijun, Li Xinrong, Xu Yingshan, Guo Hongji, Huang Zifeng, Wang Qizhang, Liu Helu, Chen Dongfeng, Zhu Meiling
Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine, Guangzhou University of Chinese Medicine, Guangdong, China.
The Research Center of Basic Integrative Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China.
Front Neurol. 2021 Sep 10;12:719354. doi: 10.3389/fneur.2021.719354. eCollection 2021.
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. 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. 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. 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.
电针（EA）治疗缺血性中风的复杂分子机制仍不清楚。最近，更多证据揭示了微小RNA（miRNA）-信使核糖核酸（mRNA）网络在缺血性中风中的重要作用。然而，目前仍缺乏对EA在缺血性中风中调控的新型关键基因、miRNA及miRNA-mRNA网络的系统分析。我们建立了大脑中动脉闭塞（MCAO）小鼠模型，并对缺血性中风小鼠进行EA治疗。应用行为测试和梗死面积测量来评估EA治疗的效果。然后，我们进行RNA测序以分析EA组和对照组之间的差异表达基因（DEG）及功能富集情况。此外，构建了蛋白质-蛋白质相互作用（PPI）网络，并通过Cytoscape筛选出枢纽基因。通过miRTarBase预测上游miRNA。然后通过逆转录定量聚合酶链反应（RT-qPCR）验证枢纽基因和预测的miRNA作为关键生物标志物。最后，构建miRNA-mRNA网络以探索EA在缺血性中风中的潜在机制。我们的分析表明，EA治疗可显著减轻MCAO模型小鼠患侧肢体的神经功能缺损并减小梗死面积。在EA组和对照组之间共鉴定出174个显著的DEG，其中包括53个上调基因和121个下调基因。功能富集分析表明，这些DEG与叉头框O（FOXO）信号通路、核因子κB（NF-κB）信号通路、T细胞受体信号通路及其他重要通路相关。根据度值法，将度值最高的前10个基因鉴定为枢纽基因，但根据RT-qPCR仅7个基因被验证为关键基因。预测有12个上游miRNA靶向这7个关键基因。然而，只有4个miRNA显著上调并表明EA治疗具有良好效果。最后，对结果的综合分析确定了miR-425-5p-Cdk1、mmu-miR-1186b-Prc1、mmu-miR-434-3p-Prc1和mmu-miR-453-PrcmiRNA-mRNA网络为受EA调控且与缺血性中风相关的关键网络。这些网络可能主要发生在缺血性中风中受EA调控的神经元细胞中。总之，我们的研究确定了关键的DEG、miRNA及miRNA-mRNA调控网络，这可能有助于促进对EA治疗缺血性中风作用的分子机制的理解。
