DataSheet_1_Developmental Regulation and Functional Prediction of microRNAs in an Expanded Fasciola hepatica miRNome.docx

The liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analyzed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology.

肝片形吸虫（Fasciola hepatica）是全球范围内严重危害养殖反刍动物健康与生产性能的病原，同时也是威胁人类健康的人畜共患寄生虫。尽管学界迫切需要加速开发针对该病原体的新型药物与疫苗疗法，但目前我们对肝片形吸虫的生物学特性及其与宿主的互作机制仍知之甚少。非编码RNA（noncoding RNAs），包括微小RNA（miRNA），是所有真核生物转录调控的关键分子，因此解析miRNA的生物学功能可从系统层面揭示生物体的整体功能。此前已有四项研究报道了肝片形吸虫的最多89条成熟miRNA序列，但本研究数据显示，该结果并未完整涵盖该物种的miRNA组（miRNome）。本研究在既往研究基础上，首次对肝片形吸虫多个生活史阶段的miRNA进行测序，涵盖成虫、新脱囊幼虫（newly excysted juvenile，NEJ）、囊蚴以及成虫来源的细胞外囊泡（extracellular vesicles，EVs）。实验共检测到61条新增的高可信度miRNA，其中多数未在其他物种中被报道，由此将肝片形吸虫的miRNA组扩增至150条成熟序列。我们通过定量聚合酶链反应（qPCR）实验，首次绘制了该物种miRNA在囊蚴、新脱囊幼虫、成虫及成虫来源细胞外囊泡中的发育表达谱。多数miRNA在囊蚴中表达量最高，且在不同生活史阶段中可区分出至少6个独特的表达簇。我们对细胞内miRNA进行功能分析，通过与肝片形吸虫组织转录组中呈负相关表达的靶mRNA进行关联，揭示了其与关键毒力转录本（包括组织蛋白酶蛋白酶）以及调控寄生虫生长、发育和运动的神经肌肉基因的调控互作关系。本研究还将28条成虫来源的细胞外囊泡miRNA与片形吸虫感染宿主后，反刍动物淋巴结、外周血单个核细胞（peripheral blood mononuclear cell，PBMC）及肝脏组织转录组中397个下调的宿主基因进行关联。这些下调基因涉及信号转导、免疫及代谢通路，进一步佐证了片形吸虫病（fasciolosis）中基于miRNA的免疫抑制机制。本研究数据拓展了我们对肝片形吸虫miRNA组的认知，首次提供了该物种miRNA发育调控的相关数据，并为肝片形吸虫miRNA生物学的功能基因组学研究提供了一系列可验证的假说。