双瓶选择饮酒致酒精依赖小鼠海马中长链非编码RNA的表达谱分析

Expression profile analysis of long non⁃coding RNAs in hippocampus of alcohol⁃dependent mice induced by double⁃bottle selective drinking

目的对双瓶选择饮酒致酒精依赖小鼠海马中的长链非编码RNA(long non-coding RNA,lncRNA)表达谱进行分析。方法通过双瓶选择饮酒法建立酒精依赖小鼠模型,同时设对照组(饮水)。选取酒精组酒精偏好率>60%且酒精饮用量>10 g/(kg·24 h)的雄性小鼠3只,随机抽取对照组雄性小鼠3只,分离双侧海马脑组织,液氮保存。采用Agilent084388芯片对小鼠海马脑组织RNA样本lncRNA和mRNA进行测序分析,lncRNA表达谱芯片(ncRNA microarray)检测样本中lncRNA的差异表达情况。基因功能(Gene Ontology,GO)和信号通路数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析差异表达lncRNA可能涉及的生物学过程及参与的信号通路。Pearson相关分析预测与每个差异表达lncRNA共表达的编码基因,超几何分布检验法计算每个对应转录因子条目中差异基因富集的显著性,Cytoscape软件绘制可视化网络图。结果与对照组比较,酒精组小鼠海马组织差异表达的lncRNA共855个(FC≥2.0,P<0.05),337个显著上调,上调幅度最大的为NONMMUT025786.2和NONMMUT072246.2;518个显著下调,下调幅度最大的为NONMMUT113098.1和NONMMUT076455.1。两组小鼠差异表达mRNA共361个(FC≥2.0,P<0.05),271个显著上调,Upf3b和Zfp943上调幅度最大;90个显著下调,Adamts13和Ift27下调幅度最大。差异表达的lncRNA以细胞表面正向调控、GTP酶活性、细胞囊泡运输差异最明显;其主要参与的信号通路有丙酸酯代谢通路、牛磺酸代谢通路、细胞外基质受体和AMPK信号通路。最富集的转录因子为FOXL1和LHX3,分别有25和21个对应共表达基因。结论通过高通量基因表达谱芯片分析,获得了lncRNA和mRNA可能的关键调控位点。本研究为海马脑区酒精依赖相关分子机制的研究提供了实验依据。

Objective To analyze the expression profiles of long non-coding RNAs(lncRNA)in hippocampus of alcoholdependent mice induced by double-bottle selective drinking.Methods The alcohol-dependent mouse model was established by double-bottle selective drinking method,and the control group was set up(drinking water).Three male mice with alcohol preferencemorethan 60%andalcoholconsumptionmorethan 10g/(kg·24h)in alcohol group and random three male micein control group were selected,of which bilateral hippocampal brain tissues were isolated and stored in liquid nitrogen.LncRNA and mRNA of mouse hippocampal brain tissue RNA samples were sequenced by using Agilent-084388 microarray,and the differential expression of lncRNA in samples was detected by using ncRNA microarray.The biological processes andsignaling pathways involved in differential expression of lncRNA were clustered and enriched by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Pearson correlation analysis was used to predict the coding genes co-expressed by each differentially expressed lncRNA.Hypergeometric distribution test was used to calculate the significance of differential gene enrichment in each corresponding transcription factor item,and Cytoscape software was used to draw a visual network diagram.Results Compared with the control group,totally 855 lncRNAs(FC≥2.0,P<0.05)were differentially expressed in the hippocampus of mice in alcohol group,of which 337 lncRNAs were up-regulated significantly,with NONMMUT025786.2 and NONMMUT072246.2 being the most up-regulated,and 518 significant downward adjustments were observed,with the largest downward adjustments being NONMMUT113098.1 and NONMMUT076455.1.There were 361 mRNAs differentially expressed in the two groups(FC≥2.0,P<0.05)with 271 mRNAs up-regulated significantly and 90 significant downward adjustments,among which,the most obvious up-regulated were Upf3b and Zfp943,and Adamts 13 and Ift 27 showed the largest downward adjustments.The differential expression of lncRNAs was most obvious in the positive regulation of cell surface,GTPase activity and cell vesicle transport;The main signaling pathways involved were propanoate metabolism,taurine metabolism,extracellular matrix receptor interaction and AMPK signaling pathway.The most abundant transcription factors were FOXL1 and LHX3,with 25 and 21 corresponding co-expressed genes,respectively.Conclusion Through high-throughput gene expression profile microarrayan alysis,the possible key regulatory sites of lncRNAs and mRNAs were obtained,which provided experimental basis for research of the molecular mechanism of alcohol dependence in the hippocampus.