Abstract：
This study aims to explore the molecular mechanisms of miR-206-3p in regulating Hsp90aa1 and its involvement in the central nervous system (CNS) injury in heat stroke. Weighted gene co-expression network analysis (WGCNA) was performed on the GSE64778 dataset of heat stroke to identify module genes most closely associated with disease characteristics. Through the selection of key genes and predicting upstream miRNAs using RNAInter and miRWalk databases, the regulatory relationship between miR-206-3p and Hsp90aa1 was determined. Through in vitro experiments, various methods, including bioinformatics analysis, dual-luciferase reporter gene assay, RIP experiment, and RNA pull-down experiment, were utilized to validate this regulatory relationship. Furthermore, functional experiments, including CCK-8 assay to test neuron cell viability and flow cytometry to assess neuron apoptosis levels, confirmed the role of miR-206-3p. Transmission electron microscopy, real-time quantitative PCR, DCFH-DA staining, and ATP assay were employed to verify neuronal mitochondrial damage. Heat stroke rat models were constructed, and mNSS scoring and cresyl violet staining were utilized to assess neural functional impairment. Biochemical experiments were conducted to evaluate inflammation, brain water content, and histopathological changes in brain tissue using H&E staining. TUNEL staining was applied to detect neuronal apoptosis in brain tissue. RT-qPCR and Western blot were performed to measure gene and protein expression levels, further validating the regulatory relationship in vivo. Bioinformatics analysis indicated that miR-206-3p regulation of Hsp90aa1 may be involved in CNS injury in heat stroke. In vivo, animal experiments demonstrated that miR-206-3p and Hsp90aa1 co-localized in neurons of the rat hippocampal CA3 region, and with prolonged heat stress, the expression of miR-206-3p gradually increased while the expression of Hsp90aa1 gradually decreased. Further in vitro cellular mechanism validation and functional experiments confirmed that miR-206-3p could inhibit neuronal cell viability and promote apoptosis and mitochondrial damage by targeting Hsp90aa1. In vivo, experiments confirmed that miR-206-3p promotes CNS injury in heat stroke. This study revealed the regulatory relationship between miR-206-3p and Hsp90aa1, suggesting that miR-206-3p could regulate the expression of Hsp90aa1, inhibit neuronal cell viability, and promote apoptosis, thereby contributing to CNS injury in heat stroke.
摘要：
本研究旨在探讨miR-206-3p调控Hsp90aa1及其参与中暑中枢神经系统(CNS)损伤的分子机制。对中暑的GSE64778数据集进行加权基因共表达网络分析（WGCNA），以鉴定与疾病特征最密切相关的模块基因。通过选择关键基因并使用RNAInter和miRWalk数据库预测上游miRNA，确定miR-206-3p与Hsp90aa1之间的调控关系.通过体外实验,各种方法,包括生物信息学分析，双荧光素酶报告基因测定,RIP实验,和RNA下拉实验，被用来验证这种监管关系。此外，功能实验，包括CCK-8测定神经元细胞活力和流式细胞术评估神经元凋亡水平，证实了miR-206-3p的作用。透射电子显微镜，实时定量PCR,DCFH-DA染色,和ATP试验用于验证神经元线粒体损伤。建立中暑大鼠模型,mNSS评分和甲酚紫染色用于评估神经功能缺损。进行生化实验以评估炎症，脑含水量,和脑组织的组织病理学变化使用H&E染色。应用TUNEL染色检测脑组织中神经元凋亡。RT-qPCR和Westernblot检测基因和蛋白表达水平,进一步验证了体内的调控关系。生物信息学分析表明miR-206-3p调控Hsp90aa1可能参与了中暑中枢神经系统的损伤。在体内,动物实验表明，miR-206-3p和Hsp90aa1共定位在大鼠海马CA3区的神经元中，长期的热应力，miR-206-3p的表达逐渐升高,而Hsp90aa1的表达逐渐降低。进一步的体外细胞机制验证和功能实验证实，miR-206-3p可以通过靶向Hsp90aa1抑制神经元细胞活力，促进细胞凋亡和线粒体损伤。在体内,实验证实miR-206-3p促进中暑中枢神经系统损伤。本研究揭示了miR-206-3p与Hsp90aa1的调控关系,提示miR-206-3p可以调控Hsp90aa1的表达,促进细胞凋亡，从而导致中暑中枢神经系统损伤。
