Supplementary Material for: The Exosomal MicroRNA Profile Is Responsible for the Mesenchymal Stromal Cell Transplantation-Induced Improvement of Functional Recovery after Stroke in Rats

Introduction: The mechanism through which mesenchymal stromal cells (MSCs) enhance functional recovery in experimental models of stroke remains to be elucidated. This study was carried out to determine the microRNA (miRNA) profile elicited in response to MSC transplantation after stroke. Methods: This was an in vivo study on the effect of MSC transplantation on the exosomal miRNA profile in a rat model of middle cerebral artery occlusion (MCAO)-induced stroke. Eighteen male Sprague-Dawley rats were subjected to MCAO surgery (model group), and half received a transplantation of MSCs (model + MSC group) isolated from rat bone marrow. A sham-operated group (Sham) was included as a control. After 7 days, the volume of the brain lesion and severity of the functional impairments were measured. Exosomes were isolated from blood plasma samples for miRNA transcriptome analysis by Illumina sequencing. Results: The MCAO surgery successfully induced infarcts and neurological deficits in the rats, whereas the MSC transplantation significantly repaired these impairments. Illumina sequencing identified 764 known miRNAs, including 135 that were differentially expressed in common between the model + MSC and model, model and Sham, and model + MSC and Sham groups, respectively. Gene Ontology enrichment analysis revealed that the target genes of these miRNAs were associated with biological processes relevant to learning or memory and the development of the central and peripheral nervous systems. Pathway enrichment analysis identified a cluster of miRNAs (e.g., rno-miR-19b-3p, rno-miR-204-3p, rno-miR-125a-5p, rno-miR-672-3p, and rno-miR-667-3p) to be significantly related to the Janus kinase-signal transducer and activator of transcription, mechanistic target of rapamycin, phosphoinositide 3-kinases–Akt, and insulin signaling pathways via their control of their gene targets. Conclusion: We confirmed that MSC transplantation repaired stroke-induced functional impairments in rats by regulating various pathways associated with nervous system protection and MSC differentiation through the deregulation of exosomal miRNAs.

引言：间充质基质细胞（mesenchymal stromal cells, MSCs）改善脑卒中实验模型中功能恢复的具体机制仍有待阐明。本研究旨在探究脑卒中后接受MSCs移植所诱导的微小RNA（microRNA, miRNA）表达谱。
方法：本研究为体内实验，聚焦MSCs移植对大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）诱导的脑卒中大鼠模型中外泌体miRNA表达谱的影响。将18只雄性斯普拉-道来（Sprague-Dawley, SD）大鼠实施MCAO手术以构建脑卒中模型，随机分为模型组；其中半数大鼠接受源自大鼠骨髓的MSCs移植，设为模型+MSCs组；另设假手术组（Sham）作为对照。术后7天，检测各组大鼠的脑梗死体积与神经功能损伤严重程度。采集血浆样本分离外泌体，通过Illumina测序（Illumina sequencing）开展miRNA转录组分析。
结果：MCAO手术成功在大鼠体内诱导出脑梗死与神经功能缺损，而MSCs移植可显著改善此类损伤。Illumina测序共鉴定出764种已知miRNA，其中分别有135种在模型+MSCs组与模型组、模型组与假手术组、模型+MSCs组与假手术组间存在共同差异表达。基因本体（Gene Ontology, GO）富集分析显示，上述miRNA的靶基因参与与学习记忆、中枢及外周神经系统发育相关的生物学过程。通路富集分析显示，包括rno-miR-19b-3p、rno-miR-204-3p、rno-miR-125a-5p、rno-miR-672-3p及rno-miR-667-3p在内的一系列miRNA，可通过调控靶基因，显著参与贾纳斯激酶-信号转导与转录激活因子（JAK-STAT）、雷帕霉素靶蛋白（mTOR）、磷脂酰肌醇3-激酶-Akt（PI3K-Akt）及胰岛素信号通路。
结论：本研究证实，MSCs移植可通过调控外泌体miRNA的表达，改变一系列与神经系统保护及MSCs分化相关的通路，从而改善脑卒中诱导的大鼠功能损伤。