基于RNA-seq的脓毒症相关性脑病小鼠海马转录组分析及竞争性内源性RNA调控网络的构建  

Transcriptomic analyses in hippocampus based on RNA-seq and construction of a competing endogenous RNA regulatory network in mice with sepsis-associated encephalopathy

作　　者：张英立[1] 于明懂[1] 刘乘麟 李佩 王慧星[4] 张婧[1] 魏立国[1] 于泳浩[3] 谢克亮[3] 卢悦淳[1] Zhang Yingli;Yu Mingdong;Liu Chenglin;Li Pei;Wang Huixing;Zhang Jing;Wei Liguo;Yu Yonghao;Xie Keliang;Lu Yuechun(Department of Anesthesiology,The Second Hospital of Tianjin Medical University,Tianjin 30021l,China;Department of Anesthesiology,Tianjin Hospital,Tianjin 300211,China;Department of Anesthesiology,Tianjin Medical University General Hospital,Tianjin Research Institute of Anesthesiology,Tianjin 300052,China;Pain Management Center,The Second Hospital of Tianjin Medical University,Tianjin 300211,China)

机构地区：[1]天津医科大学第二医院麻醉科,天津300211 [2]天津市天津医院麻醉科,天津300211 [3]天津医科大学总医院麻醉科、天津市麻醉学研究所,天津300052 [4]天津医科大学第二医院疼痛治疗中心,天津300211

出　　处：《中华麻醉学杂志》2023年第9期1117-1123,共7页Chinese Journal of Anesthesiology

基　　金：天津医科大学第二医院青年科研基金项目(2020ydey11);2023天津麻醉科研发展计划(TJMZ2023-002);天津市卫生健康科技项目(ZC20058)。

摘　　要：目的采用转录组测序技术(RNA-seq)筛选脓毒症相关性脑病(SAE)小鼠海马差异表达的长链非编码RNA(lncRNA)及mRNA,构建竞争性内源性RNA(ceRNA)调控网络。方法清洁级健康雄性C57BL/6小鼠10只,6~8周龄,体质量20~25 g,采用随机数字表法分为2组(n=5):假手术组(Sham组)和脓毒症组(Sepsis组)。Sepsis组采用盲肠结扎穿孔(CLP)法制备小鼠脓毒症模型,Sham组只开腹不进行盲肠结扎和穿孔。分别于术前1 d及术后3 d时行Morris水迷宫实验和条件恐惧实验检测小鼠认知功能。随后Sham组随机处死3只小鼠,Sepsis组处死认知功能测试最差的3只小鼠,取海马组织,通过BGISEQ-500平台行转录组测序,得到差异表达的mRNA和lncRNA,测序结果通过深圳华大基因科技服务有限公司提供的Dr.Tom平台行可视化分析,利用Cytoscape软件构建ceRNA调控网络。结果与Sham组比较,Sepsis组逃避潜伏期延长,靶向限停留时间百分比和僵直时间百分比降低(P<0.05)。转录组数据分析共获得差异表达的lncRNA 62个,其中45个lncRNA表达上调,17个lncRNA表达下调;差异表达的mRNA 282个,其中173个mRNA表达上调,109个mRNA表达下调。对差异表达的mRNA进行生物学过程的GO富集分析,结果显示差异表达的mRNA参与了记忆、学习或记忆、炎症应答、衰老相关行为减退的调空、突触可塑性调节等生物学过程;对差异表达的mRNA进行KEGG通路富集分析,结果显示差异表达的mRNA富集于IL-17信号通路、TNF信号通路、NF-κB信号通路等。对差异表达的mRNA进行KDA分析,结果示Ch25h、Il6ra、Lcn2、Sgk1、Nr4a3、Osm、Saa3、Ccl7、Sqle、Dhcr24为SAE的关键基因。基于9个lncRNA、28个mRNA和134个miRNA成功构建了SAE小鼠海马的ceRNA调控网络。结论RNA-seq结果分析发现Ch25h、Il6ra、Lcn2、Sgk1、Nr4a3、Osm、Saa3、Ccl7、Sqle、Dhcr2410个mRNA以及Rian、Gm35874、Gm34347等lncRNA是调控小鼠SAE的关键基因;同时成功构建了SAE小鼠海马Objective To identify the differentially expressed long-chain non-coding RNA(lncRNA)and mRNA using ribonucleic acid sequencing(RNA-seq),and construct a competing endogenous RNA(ceRNA)regulatory network in mice with sepsis-associated encephalopathy.Methods Ten clean-grade healthy male C57BL/6 mice,aged 6-8 weeks,weighing 20-25 g,were divided into 2 groups(n=5 each)using a random number table method:sham operation group(group Sham)and sepsis group(group Sepsis).Sepsis was induced by cecal ligation and puncture(CLP)in group Sepsis,while group Sham only underwent laparotomy without CLP.Morris water maze test and contextual fear conditioning test were performed to detect the cognitive function on 1 day before CLP and 3 days after CLP.Three mice were randomly sacrificed in group Sham,and 3 mice with the worst results in the cognitive function test were sacrificed in group Sepsis.The hippocampal tissues were obtained for RNA-seq via the BGISEQ-500 platform,and the differentially expressed mRNA and lncRNA were identified.The differentially expressed mRNAs and lncRNAs were visualized and analyzed by Dr.Tom platform provided by Shenzhen BGI Technology Service Co.,Ltd.,and the ceRNA regulatory network was constructed using the online visualization tool Cytoscape software.Results Compared with group Sham,the escape latency was significantly prolonged,and the percentage of time of staying at the target quadrants and percentage of time spent freezing were decreased in group Sepsis(P<0.05).A total of 62 differentially expressed lncRNAs were obtained from RNA-seq,of which the expression of 45 lncRNAs was up-regulated and the expression of 17 lncRNAs was down-regulated.There were 282 differentially expressed mRNAs identified from RNA-seq,of which the expression of 173 mRNAs was up-regulated,and the expression of 109 mRNAs was down-regulated.Gene Ontology enrichment analysis revealed that the differentially expressed mRNAs were involved in biological processes such as memory,learning or memory,inflammatory responses,regulation of

关 键 词：脓毒症相关性脑病 基因表达谱 高通量核苷酸序列分析 RNA 

分 类 号：R459.7[医药卫生—急诊医学]