DataSheet1_Identification of programmed cell death-related gene signature and associated regulatory axis in cerebral ischemia/reperfusion injury.xlsx

Background: Numerous studies have suggested that programmed cell death (PCD) pathways play vital roles in cerebral ischemia/reperfusion (I/R) injury. However, the specific mechanisms underlying cell death during cerebral I/R injury have yet to be completely clarified. There is thus a need to identify the PCD-related gene signatures and the associated regulatory axes in cerebral I/R injury, which should provide novel therapeutic targets against cerebral I/R injury.
Methods: We analyzed transcriptome signatures of brain tissue samples from mice subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and matched controls, and identified differentially expressed genes related to the three types of PCD(apoptosis, pyroptosis, and necroptosis). We next performed functional enrichment analysis and constructed PCD-related competing endogenous RNA (ceRNA) regulatory networks. We also conducted hub gene analysis to identify hub nodes and key regulatory axes.
Results: Fifteen PCD-related genes were identified. Functional enrichment analysis showed that they were particularly associated with corresponding PCD-related biological processes, inflammatory response, and reactive oxygen species metabolic processes. The apoptosis-related ceRNA regulatory network was constructed, which included 24 long noncoding RNAs (lncRNAs), 41 microRNAs (miRNAs), and 4 messenger RNAs (mRNAs); the necroptosis-related ceRNA regulatory network included 16 lncRNAs, 20 miRNAs, and 6 mRNAs; and the pyroptosis-related ceRNA regulatory network included 15 lncRNAs, 18 miRNAs, and 6 mRNAs. Hub gene analysis identified hub nodes in each PCD-related ceRNA regulatory network and seven key regulatory axes in total, namely, lncRNA Malat1/miR-181a-5p/Mapt, lncRNA Malat1/miR-181b-5p/Mapt, lncRNA Neat1/miR-181a-5p/Mapt, and lncRNA Neat1/miR-181b-5p/Mapt for the apoptosis-related ceRNA regulatory network; lncRNA Neat1/miR-181a-5p/Tnf for the necroptosis-related ceRNA regulatory network; lncRNA Malat1/miR-181c-5p/Tnf for the pyroptosis-related ceRNA regulatory network; and lncRNAMalat1/miR-181a-5p for both necroptosis-related and pyroptosis-related ceRNA regulatory networks.
Conclusion: The results of this study supported the hypothesis that these PCD pathways (apoptosis, necroptosis, pyroptosis, and PANoptosis) and crosstalk among them might be involved in ischemic stroke and that the key nodes and regulatory axes identified in this study might play vital roles in regulating the above processes. This may offer new insights into the potential mechanisms underlying cell death during cerebral I/R injury and provide new therapeutic targets for neuroprotection.

背景：大量研究表明，程序性细胞死亡（programmed cell death, PCD）通路在脑缺血/再灌注（cerebral ischemia/reperfusion, I/R）损伤中发挥至关重要的作用。然而，脑I/R损伤过程中细胞死亡的具体分子机制尚未完全阐明。因此，亟需明确脑I/R损伤中与PCD相关的基因特征及其调控轴，以期为脑I/R损伤提供全新的治疗靶点。
方法：本研究对接受大脑中动脉闭塞/再灌注（middle cerebral artery occlusion/reperfusion, MCAO/R）造模的小鼠脑组织样本及匹配对照样本的转录组特征进行分析，筛选出与三类PCD（凋亡、焦亡、坏死性凋亡）相关的差异表达基因。随后开展功能富集分析，并构建PCD相关内源竞争RNA（competing endogenous RNA, ceRNA）调控网络。此外，通过核心基因分析识别核心节点与关键调控轴。
结果：共筛选得到15个PCD相关基因。功能富集分析显示，这些基因显著富集于PCD相关生物学过程、炎症反应以及活性氧代谢过程。本研究构建了凋亡相关ceRNA调控网络，包含24个长链非编码RNA（long noncoding RNA, lncRNAs）、41个微小RNA（microRNA, miRNAs）与4个信使RNA（messenger RNA, mRNAs）；坏死性凋亡相关ceRNA调控网络包含16个lncRNAs、20个miRNAs与6个mRNAs；焦亡相关ceRNA调控网络包含15个lncRNAs、18个miRNAs与6个mRNAs。核心基因分析识别出各PCD相关ceRNA调控网络中的核心节点，共得到7条关键调控轴：凋亡相关ceRNA调控网络中的lncRNA Malat1/miR-181a-5p/Mapt、lncRNA Malat1/miR-181b-5p/Mapt、lncRNA Neat1/miR-181a-5p/Mapt及lncRNA Neat1/miR-181b-5p/Mapt；坏死性凋亡相关ceRNA调控网络中的lncRNA Neat1/miR-181a-5p/Tnf；焦亡相关ceRNA调控网络中的lncRNA Malat1/miR-181c-5p/Tnf；以及同时参与坏死性凋亡与焦亡相关ceRNA调控网络的lncRNA Malat1/miR-181a-5p。
结论：本研究结果支持以下假说：上述PCD通路（凋亡、坏死性凋亡、焦亡及PANoptosis（泛凋亡））及其相互串扰可能参与缺血性脑卒中的发生发展，本研究鉴定的核心节点与调控轴可能在调控上述过程中发挥关键作用。本研究可为阐明脑I/R损伤中细胞死亡的潜在机制提供新视角，并为神经保护治疗提供全新的靶点。