Table_6_Toxocara canis Differentially Affects Hepatic MicroRNA Expression in Beagle Dogs at Different Stages of Infection.xls

Toxocara canis is a neglected zoonotic parasite, which threatens the health of dogs and humans worldwide. The molecular mechanisms that underlie the progression of T. canis infection remain mostly unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been identified in T. canis; however, the regulation and role of miRNAs in the host during infection remain incompletely understood. In this study, we determined hepatic miRNA expression at different stages of T. canis infection in beagle dogs. Individual dogs were infected by 300 embryonated T. canis eggs, and their livers were collected at 12 hpi (hours post-infection), 24 hpi, and 36 dpi (days post-infection). The expression profiles of liver miRNAs were determined using RNA-sequencing. Compared to the control groups, 9, 16, and 34 differentially expressed miRNAs (DEmiRNAs) were detected in the livers of infected dogs at the three infection stages, respectively. Among those DEmiRNAs, the novel-294 and cfa-miR-885 were predicted to regulate inflammation-related genes at the initial stage of infection (12 hpi). The cfa-miR-1839 was predicted to regulate the target gene TRIM71, which may influence the development of T. canis larvae at 24 hpi. Moreover, cfa-miR-370 and cfa-miR-133c were associated with immune response at the final stage of infection (36 dpi). Some immunity-related Gene Ontology terms were enriched particularly at 24 hpi. Likewise, Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that many significantly enriched pathways were involved in inflammation and immune responses. The expression level of nine DEmiRNAs was validated using quantitative real-time PCR (qRT-PCR). These results show that miRNAs play critical roles in the pathogenesis of T. canis during the hepatic phase of parasite development. Our data provide fundamental information for further investigation of the roles of miRNAs in the innate/adaptive immune response of dogs infected by T. canis.

犬弓首蛔虫（Toxocara canis）是一种被忽视的人兽共患寄生虫，在全球范围内威胁犬类与人类的健康。其感染进程的分子机制迄今尚未完全阐明。微小RNA（microRNAs, miRNAs）是一类已在犬弓首蛔虫中被鉴定出的小型非编码RNA，但其在感染宿主过程中的调控机制与功能仍未被完全厘清。
本研究针对比格犬感染犬弓首蛔虫后的不同阶段，检测了其肝脏组织内的miRNA表达谱。实验中，每只实验犬经口接种300枚犬弓首蛔虫胚化虫卵，并分别于感染后12小时（12 hpi, hours post-infection）、24小时（24 hpi）以及感染后36天（36 dpi, days post-infection）采集肝脏组织样本。
通过RNA测序（RNA-sequencing）分析肝脏组织的miRNA表达特征。与对照组相比，在三个感染时间点的感染犬肝脏组织中，分别检测到9种、16种和34种差异表达miRNA（differentially expressed miRNAs, DEmiRNAs）。
在上述差异表达miRNA中，novel-294与cfa-miR-885被预测可在感染初期（12 hpi）调控炎症相关基因；cfa-miR-1839的靶基因为TRIM71，该基因可能在感染后24小时（24 hpi）影响犬弓首蛔虫幼虫的发育；此外，cfa-miR-370与cfa-miR-133c与感染末期（36 dpi）的免疫应答相关。
部分免疫相关的基因本体（Gene Ontology, GO）富集条目尤其在24 hpi时显著富集。同样，京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析显示，诸多显著富集的通路均与炎症及免疫应答密切相关。
研究通过实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）验证了9种差异表达miRNA的表达水平。上述结果表明，miRNA在犬弓首蛔虫发育的肝脏阶段的致病过程中发挥关键作用。
本研究的数据为后续探究miRNA在犬感染犬弓首蛔虫后天然/适应性免疫应答中的功能提供了基础依据。