The 651 differentially expressed miRNAs.

Long non-coding RNAs (lncRNAs) are among the most abundant types of non-coding RNAs in the genome and exhibit particularly high expression levels in the brain, where they play crucial roles in various neurophysiological and neuropathological processes. Although ischemic stroke is a complex multifactorial disease, the involvement of brain-derived lncRNAs in its intricate regulatory networks remains inadequately understood. In this study, we established a cerebral ischemia-reperfusion injury model using middle cerebral artery occlusion (MCAO) in male Sprague-Dawley rats. High-throughput sequencing was performed to profile the expression of cortical lncRNAs post-stroke, with subsequent validation using RT-PCR and qRT-PCR. Among the 31,183 lncRNAs detected in the rat cerebral cortex, 551 were differentially expressed between the MCAO and sham-operated groups in the ipsilateral cortex (fold change ≥2.0, P < 0.05). An integrated analysis of the 20 most abundant and significantly differentially expressed lncRNAs (DELs) identified 25 core cytoplasmic DELs, which were used to construct an interaction network based on their targeting relationships. This led to the establishment of a comprehensive lncRNA-miRNA-mRNA regulatory network comprising 12 lncRNAs, 16 sponge miRNAs, and 191 target mRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that differentially expressed mRNAs (DEmRNAs) were significantly enriched in stroke-related pathways. Our analysis predicted four key lncRNAs, four miRNAs, and eleven crucial mRNAs involved in post-transcriptional regulation through competing endogenous RNA (ceRNA) mechanisms. These molecules were shown to participate extensively in post-stroke processes, including angiogenesis, axonal regeneration, inflammatory responses, microglial activation, blood-brain barrier (BBB) disruption, apoptosis, autophagy, ferroptosis, and thrombocytopenia. These findings highlight the role of lncRNAs as multi-level regulators in the complex network of post-stroke mechanisms, providing novel insights into the pathophysiological processes of stroke.

长链非编码RNA（long non-coding RNAs，lncRNAs）是基因组中丰度最高的非编码RNA类型之一，在脑组织中呈现极高的表达水平，并在多种神经生理及神经病理过程中发挥关键调控作用。尽管缺血性脑卒中是一种多因素复杂疾病，但脑源性lncRNA在其复杂调控网络中的参与机制仍未得到充分阐明。本研究采用雄性斯普拉格-道利大鼠（Sprague-Dawley rats）构建大脑中动脉闭塞（middle cerebral artery occlusion，MCAO）诱导的脑缺血再灌注损伤模型，通过高通量测序技术对卒中后脑皮层lncRNA的表达谱进行分析，并采用逆转录聚合酶链反应（reverse transcription polymerase chain reaction，RT-PCR）与实时定量逆转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction，qRT-PCR）进行验证。在大鼠大脑皮层检测到的31183个lncRNA中，缺血侧皮层的MCAO组与假手术组间共有551个lncRNA存在差异表达（倍数变化≥2.0，P<0.05）。对丰度最高且差异显著的20个差异表达lncRNA（differentially expressed lncRNAs，DELs）进行整合分析，筛选得到25个核心胞质差异表达lncRNA，并基于其靶向调控关系构建相互作用网络。据此构建了包含12个lncRNA、16个海绵miRNA以及191个靶mRNA的完整lncRNA-miRNA-mRNA调控网络。基因本体论（Gene Ontology，GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析结果显示，差异表达mRNA（differentially expressed mRNAs，DEmRNAs）显著富集于卒中相关通路中。本研究通过内源竞争RNA（competing endogenous RNA，ceRNA）机制预测得到4个关键lncRNA、4个miRNA以及11个核心靶mRNA，参与卒中后的转录后调控过程。这些分子广泛参与卒中后的多种病理生理过程，包括血管生成、轴突再生、炎症反应、小胶质细胞活化、血脑屏障（blood-brain barrier，BBB）破坏、细胞凋亡、自噬、铁死亡以及血小板减少症。本研究结果证实了lncRNA作为多层级调控因子在卒中后复杂机制网络中的重要作用，为阐明脑卒中的病理生理过程提供了全新的研究视角。