日本血吸虫m6A修饰的性别相关circRNA鉴定  

Identification of gender associated m6A modified circRNA in Schistosoma japonicum

作　　者：刘华熳 Bikash Giri 方传涛 郑亚萌 吴慧欣 曾敏浩 李姗 程国锋 LIU Huaman;Bikash Giri;FANG Chuantao;ZHENG Yameng;WU Huixin;ZENG Minhao;LI Shan;CHENG Guofeng(School of Medicine,Tongji University,Shanghai 200331,China;Institute for Infectious Diseases and Vaccine Development,Tongji University,Shanghai 200331,China;Shanghai Tenth People’s Hospital,Tongji University,Shanghai 200072,China;Jiangsu University of Science and Technology,Zhenjiang 212100,Jiangsu,China;Medical College of Jiujiang University,Jiujiang 332005,Jiangxi,China)

机构地区：[1]同济大学医学院,上海200331 [2]同济大学传染病和疫苗研究所,上海200331 [3]同济大学附属第十人民医院,上海200072 [4]江苏科技大学,镇江212100 [5]九江学院医学院,江西九江332005

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第5期552-558,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家重点研发计划政府间重点专项(2021YFE0191600);江西省自然科学基金(20212BAB215031)。

摘　　要：目的研究日本血吸虫雌雄成虫环状RNA(circRNA)的N6⁃腺苷酸甲基化(m6A)修饰,并对circRNA可能调控的miRNA进行预测。方法取(100±2)条日本血吸虫尾蚴,经腹部皮肤感染小鼠20 min,28 d后麻醉小鼠,用肝门静脉灌洗法收集虫体,分离雌雄虫,提取雌雄虫总RNA,分别取0.5μg和1.0μg点样在尼龙膜上,转移至紫外交联仪中,依次与m6A抗体(1∶1000)和HRP标记的二抗(1∶5000)孵育,进行斑点杂交。取雌雄虫总RNA各1μg,应用RNA甲基化共沉淀试剂盒进行免疫沉淀分离,RNA建库试剂盒进行建库,生物分析仪质量检测后在高通量测序仪上采用150 bp双端模式进行测序。应用Cutadapt软件(v1.9.3)除去序列中的接头与低质量序列,获得高质量序列。使用Bowtie2软件将序列与血吸虫基因组(Sja_WBPS14)匹配,以Find_circ软件识别序列中的circRNA,MACS软件识别序列中的甲基化位点。利用Heatmap2软件对差异表达的circRNA进行聚类分析,对差异有统计学意义的m6A修饰的circRNA进行来源基因的基因本体论(GO)富集分析。利用DiffReps软件分析m6A修饰的circRNA在雌雄虫的差异情况。通过RNAhybrid软件预测与circRNA相互作用的miRNA,将miRNA反转录后进行qPCR分析,2-ΔCt法计算miRNA相对表达水平。结果斑点杂交结果显示,日本血吸虫总RNA存在m6A甲基化修饰,且随着RNA量的增多,m6A甲基化信号趋于增强。高通量测序结果显示,日本血吸虫高质量序列占比高于99.9%,RNA富集与文库构建效果良好。来源于重叠区的circRNA占49%,其次是外显子(占29%)和基因间(占12%),来源于内含子(占2%)和反义链(占8%)的circRNA较少。雄虫中高富集的circRNA来源的基因可能与细胞代谢进程、细胞成分组织生物发生和应激等相关,雌虫中高富集的circRNA来源的基因可能参与细胞分子结合等过程。雌虫高丰度的m6A修饰circRNA有57个,雄虫有81个,雄虫富集的m6A修饰的circRNA数量高于雌虫(P<0.05,差异倍�Objective To explore the N6 adenylate methylation(m6A)modification of circular RNA(cir⁃cRNA)in male and female adult worms of Schistosoma japonicum,and predict its possible role in regulating miRNA.Methods S.japonicum cercariae(100±2)were used to infect mice through abdominal skin for 20 min.On 28 d post⁃infection,the mice were anesthetized to collect adult worms using hepatic portal vein perfusion method,of which the female and male worms were separated for extracting total RNA,respectively.The RNA samples of 0.5μg and 1.0μg were applied by dotting on the nylon membrane,which was transferred to a UV⁃crosslinker for dot hybridiza⁃tion through successive incubation with m6A antibody(1∶1000)and HRP labeled secondary antibody(1∶5000).Immu⁃noprecipitation separation was performed for 1μg of total RNA from both female and male worms using RNA methyla⁃tion co⁃precipitation kit.The RNA library kit was used for library construction.After quality testing with a biological analyzer,sequencing was performed using a 150 bp double⁃ended mode on a high⁃throughput sequencer.The Cutadapt software(v1.9.3)was applied to remove joints and low⁃quality sequences and obtain high⁃quality sequences.The se⁃quence was aligned with the Schistosoma genome(Sja_WBPS14)using Bowtie2 software,and the Find_circ software was used to identify circRNA,while MACS software was used to recognize methylation sites.Cluster analysis of differ⁃entially expressed circRNAs was conducted using Heatmap2 software,and gene ontology(GO)enrichment analysis was performed for the source genes of statistically significant differentiated m6A modified circRNA.DiffReps software was used to analyze the differences in m6A modified circRNA between male and female worms.Using RNAhybrid soft⁃ware,the interaction between miRNA and circRNA was predicted.The reverse⁃transcribed miRNA was analyzed by qPCR,and all the relative expression level of miRNA was calculated with 2-ΔCt method.Results The dot blot hy⁃bridization results showed tha

关 键 词：日本血吸虫 性别 m6A甲基化 circRNA MIRNA 

分 类 号：R383.24[医药卫生—医学寄生虫学]