小鼠脑缺血再灌注损伤模型中circRNA的m^(6)A修饰及表达谱分析

Analysis of m^(6)A Modification and Expression Profile of circRNA in Cerebral Ischemia-reperfusion Model

本研究通过甲基化RNA免疫沉淀测序(methylated RNA immunoprecipitation sequencing,MeRIP-seq)、转录物组测序(transcriptome sequencing,RNA-seq)及生物信息学技术,分析小鼠大脑中动脉栓塞/再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)模型中环状RNA (circular RNA,circRNA)差异表达谱和m^(6)A修饰差异表达谱,为揭示circRNA表观遗传修饰与脑缺血再灌注损伤之间关系提供科学依据。采用Longa生物学评分评价小鼠神经功能缺损,TTC染色法计算小鼠脑梗死体积,Dot印迹检测m^(6)A整体甲基化水平。结果显示,MCAO/R组与sham组相比出现严重神经损伤,Longa生物学评分为(2.75±0.25)分,MCAO/R组的梗死体积显著高于sham组,所占百分比为(27.63%±4.24%),MCAO/R组m^(6)A整体修饰水平增加。与sham组相比,MCAO/R组有1 787个差异表达的circRNAs(|log_(2)FC|> 0.58,P<0.05)。其中,852个circRNA表达显著上调,935个circRNAs表达显著下调。基因本体(gene ontgology,GO)功能分析显示,差异表达circRNAs靶基因主要参与翻译、蛋白质糖基化、激素应答、IL-6介导的信号通路、高尔基体、内质网等生物学过程;京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析发现,差异靶基因主要与N-聚糖生物合成、Wnt信号通路、胃酸分泌、谷氨酸能突触、磷脂酶D信号通路、味觉传导、非洲锥虫病、甲状腺激素合成、胰岛素分泌等代谢途径和信号通路有关。与sham组比,MCAO/R组有22个circRNAs发生m^(6)A甲基化修饰差异(|log_(2)FC|> 0.58,P<0.05)。其中,14个circRNA显著上调,8个circRNA显著下调。GO和KEGG分析显示,差异甲基化circRNA主要与胞嘧啶代谢过程、骨骼肌收缩、髓样细胞分化、O-连接蛋白质糖基化等生物学过程有关,主要富集到范科尼贫血途径通路。最后,联合表达差异和m^(6)A修饰差异进行分析,共筛选到7个共表达差异(既有m^(6)A修饰差异,又有表达差异)的circRNA,经RT-qPCR验证,这些共表达差异circRNA表达趋势与测序一致。本研究通过对sham组和MCAO/R组测序分析,筛选有差异表达的circRNA和有m^(6)A修饰差异的circRNA,表明脑缺血再灌注可引起小鼠circRNA表达谱及m^(6)A修饰谱改变,差异表达的circRNA和差异甲基化circRNA靶基因涉及多种通路,为从表观遗传水平揭示脑缺血再灌注损伤的分子机制奠定基础,为后续研究脑缺血再灌注损伤提供了潜在的靶向位点。

In this study,we performed high-throughput sequencing technology methylated RNA immunoprecipitation sequencing(MeRIP-seq),transcriptome sequencing(RNA-seq) and bioinformatics to analyze the differentially m^(6)A-methylated and differentially expressed profile of circular RNA(circRNA) in middle cerebral artery occlusion/reperfusion(MCAO/R) model,which provided some scientific evidences for revealing the relationship between RNA epigenetic modification and cerebral ischemia reperfusion injury.The neurological deficit scores of mice were evaluated by the Longa score standard.TTC staining was used to detect cerebral infarction volumes,and dot blot was used for the quantification of m^(6)A abundance.The results showed that the MCAO/R group showed severe neurological loss,and their Longa scores(2.75 ± 0.25) were significantly increased compared with that in the sham group.The percentage of cerebral infarct volumes in the MCAO/R group(27.63% ± 4.24%) was higher than that in the sham group and cerebral ischemia-reperfusion elevated global m^(6)A levels.Compared with the sham group,1787 circRNAs in the MCAO/R group were significantly changed,of which 852 circRNAs were increased and 935 circRNAs were decreased significantly(P<0.05).Gene ontology(GO) function analysis showed that differentially target genes were mainly involved in translation,protein N-linked glycosylation,response to hormone,interleukin-6-mediated signaling pathway,Golgi cisterna membrane,and integral component of endoplasmic reticulum membrane,etc.Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis found that differential circRNAs were mainly related to N-Glycan biosynthesis,Wnt signaling pathway,gastric acid secretion,glutamatergic synapse,phospholipase D signaling pathway,taste transduction,african trypanosomiasis,thyroid hormone synthesis,insulin secretion and so on.Compared with the sham group,22 m^(6)A peaks in the MCAO/R group were significantly changed,of which 14 m^(6)A peaks were significantly upregulated and 8 m^(6)A peaks were decreased significantly(P<0.05).GO and KEGG pathway analyses of the predicted target genes of the differentially m6 A-methylated circRNAs were performed.GO analysis showed that differentially m6 A-methylated target genes were mainly related to 5-methylcytosine catabolic process,chemical homeostasis within a tissue,G-protein coupled glutamate receptor binding and so on.KEGG pathway enrichment analysis indicated that the differentially m^(6)A-methylated circRNAs target genes were mainly related to Fanconi anemia pathway.By comprehensively analyzing MeRIP-seq and RNA-seq data,there were seven differentially m^(6)A-methylated and expressed circRNAs,and RT-qPCR was used to detect their expression.The target genes of the differentially m6 A-methylated and differentially expressed circRNAs were predicted.In this study,sequencing analysis of the differentially m^(6)A-methylated and differentially expressed circRNAs from sham and MCAO/R groups,and the results suggest that cerebral ischemia-reperfusion can cause changes of the circRNA expression profile and m^(6)A modification profile in mice,and target genes of differentially expressed circRNA and differentially methylated circRNA are involved in multiple functions and pathways.This study lays the foundation for revealing the molecular mechanism of cerebral ischemia-reperfusion injury from epigenetic level and provides potential target sites for subsequent research on cerebral ischemia-reperfusion injury.