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0.5 μg和1.0 μg点样在尼龙膜上，转移至紫外交联仪中，依次与m6A抗体（1∶1 000）和HRP标记的二抗（1∶5 000）孵育，进行斑点杂交。取雌雄虫总RNA各1 µg，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，差异倍数 ≥ 2）。雌雄虫中m6A修饰circRNA可潜在与多个miRNA相互作用，如Sja-Bantam、Sja-miR-307在雌虫中呈现高表达，Sja-miR-8-3p、Sja-miR-3504等在雄虫中呈现高表达。 结论 本研究获得了日本血吸虫雌雄成虫m6A修饰的circRNA表达谱，初步揭示了m6A修饰circRNA可能调控的miRNA。

Objective To explore the N6 adenylate methylation (m6A) modification of circular RNA (circRNA) 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 0.5 μg and 1.0 μg were applied by dotting on the nylon membrane, which was transferred to a UV-crosslinker for dot hybridization through successive incubation with m6A antibody (1∶1 000) and HRP labeled secondary antibody (1∶5 000). Immunoprecipitation separation was performed for 1 µg of total RNA from both female and male worms using RNA methylation 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 sequence 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 differentially 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 software, 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 hybridization results showed that there was m6A methylation modification in the total RNA of S. japonicum, and the m6A methylation signal tended to increase with the increase of RNA content. The high-throughput sequencing results showed that the proportion of high-quality sequences in S. japonicum was over 99.9%, and the RNA enrichment and library construction were effective. CircRNAs originating from overlapping regions account for 49%, followed by exons (29%) and intergenes (12%), with fewer circRNAs originating from introns (2%) and antisense chains (8%). Genes derived from highly enriched circRNAs in males may be related to cellular metabolic processes, cellular components, tissue biogenesis, and stress, while genes derived from highly enriched circRNAs in females may be involved in processes such as cell molecule binding. The number of m6A-modified circRNAs enriched in males was higher than that in females (P < 0.05, fold change ≥ 2) with 57 high abundance m6A-modified circRNAs in females and 81 in males. M6A-modified circRNAs in both male and female insects can potentially interact with multiple miRNAs, such as Sja-Bantam and Sja-miR-307, which are highly expressed in females, and Sja-miR-8-3p, Sja-miR-3504 and so on, which are highly expressed in males. Conclusion This study obtained the expression profile of m6A modified circRNA in adult worms of S. japonicum, and preliminarily revealed the miRNA that may be regulated by m6A modified circRNA.