基于RNA-seq技术分析慢性间歇性缺氧致认知障碍的机制  

Analysis of the Mechanism of Chronic Intermittent Hypoxia-induced Cognitive Impairment Based on RNA-seq Technology

作　　者：黄尹裴 刘智俐 汪鑫 刘恋[1] 李尧旭 段君[3] 张峰[4] 李兵[1] HUANG Yinpei;LIU Zhili;WANG Xin;LIU Lian;LI Yaoxu;DUAN Jun;ZHANG Feng;LI Bing(Department of Otolaryngology Head and Neck surgery,The First Affiliated Hospital of Chongqing Medical University,Chongqing,400016;Department of Oral and Maxillofacial Surgery,The First Affiliated Hospital of Chongqing Medical University,Chongqing,400016;Department of Stomatology,Children's Hospital of Chongqing Medical University,National Clinical Research Center for Child Health and Disorders,Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of Pediatrics,Chongqing,400014;Department of Otolaryngology,Children's Hospital of Chongqing Medical University,Chongqing,400014)

机构地区：[1]重庆医科大学附属第一医院耳鼻咽喉头颈外科,重庆400016 [2]重庆医科大学附属第一医院口腔颌面外科,重庆400016 [3]重庆医科大学儿童医院口腔科,国家儿童健康与疾病临床研究中心,儿童发育与疾病教育部重点实验室,重庆市儿科重点实验室,重庆400014 [4]重庆医科大学儿童医院耳鼻喉科,重庆400014

出　　处：《基因组学与应用生物学》2023年第11期1235-1246,共12页Genomics and Applied Biology

基　　金：重庆市自然科学基金面上项目(2022NSCQ-MSX0935)资助。

摘　　要：慢性间歇性缺氧(chronic intermittent hypoxia, CIH)是阻塞性睡眠呼吸暂停综合征(obstructive sleep apnea syndrome, OSA)的主要病理表现,也是OSA引起认知功能障碍的主要原因。本研究旨在基于RNA测序(RNA sequencing, RNA-seq)技术探索CIH致认知障碍在转录组层面的潜在机制。将24只SD大鼠随机分为对照组和慢性间歇性缺氧组(CIH组)。Y迷宫用于检测CIH对SD大鼠认知功能的影响。行为学测试结束后,提取大鼠海马组织总RNA进行高通量全转录组测序。R软件筛选差异表达基因(differentially expressed genes, DEGs),并获得DElncRNAs (differentially expressed lncRNAs)、 DEmiRNAs (differentially expressed miRNAs)和DEmRNAs (differentially expressed mRNAs)。基于竞争性内源RNA(competitive endogenous RNA, ceRNA)理论,用生物信息数据库RNAhybrid、 miRanda和TargetScan预测lncRNA-miRNA相互作用对和miRNA-mRNA相互作用对。用Cytoscape构建特异性ceRNA网络。RT-qPCR和Western blot在非测序大鼠海马组织中验证测序结果。Y迷宫结果表明CIH损害大鼠的空间参考学习和记忆能力。RNA-seq筛选出4个DElncRNAs、 89个DEmiRNAs和774个DEmRNAs。基于基因间相互作用,成功构建由3个DElncRNAs介导的ceRNA调控网络。GO和KEGG富集分析结果提示ceRNA网络和神经元突触活性相关。RT-qPCR和Western blot结果表明关键DEGs表达改变和RNA测序结果基本一致。本研究探讨了关键lncRNA-miRNA-mRNA ceRNA调控轴在CIH致认知障碍中潜在的生物学功能,为进一步阐明CIH致认知障碍分子机制提供了新见解。Chronic intermittent hypoxia(CIH)is the main manifestation of obstructive sleep apnea syndrome(OSA)and the main cause of OSA-induced cognitive impairment.This study aims to explore the transcriptome mechanism of CIH-induced cognitive impair-ment with RNA sequencing(RNA-seq)technology.Twenty-four SD rats were randomly divided into the control group and the chronic intermittent hypoxia group(CIH group).Y maze was used to detect the effect of CIH on cognitive function of SD rats.After the beha-vioral test,total RNA from the hippocampus of rats was extracted for high-throughput transcriptome sequencing.Differentially expressed genes(DEGs)were analyzed by R software,including DElncRNAs(differentially expressed IncRNAs),DEmiRNAs(differentially expressed miRNAs),and DEmRNAs(differentially expressed mRNAs).Based on the competitive endogenous RNA(ceRNA)mechanism,IncRNA-miRNA interaction pairs and miRNA-mRNA interaction pairs were predicted by bioinformatics databases RNA hybrid,miRanda,and TargetScan.Cytoscape was used to construct the specific ceRNA networks.Sequencing results were validated in nonsequenced rat hippocampus tissue by RT-qPCR and Western blot.The results of Y maze showed that CIH-exposed rats showed a decrease in spatial reference learning and memory.RNA-seq detected 4 DElncRNAs,89 DEmiRNAs,and 774 DEmRNAs.Three DElncRNAs-mediated ceRNA networks were constructed based on gene interaction pairs.GO and KEGG enrichment analysis suggested that the ceRNA network was related to neuronal synaptic activity.RT-qPCR and Western blot results showed that the expression changes of key DEGs were basically consistent with the results of RNA-seq.This study explores the potential biological function of the key lncRNA-miRNA-mRNA ceRNA regulatory axes in CIH-induced cognitive impairment.It would provide new insights for further elucidating the transcriptome level mechanism of CIH-induced cognitive impairment.

关 键 词：慢性间歇性缺氧 认知障碍 RNA测序(RNA-seq) lncRNA ceRNA网络 

分 类 号：R766[医药卫生—耳鼻咽喉科]