背景:细粒棘球蚴原头节(PSC)的囊化是继发包虫在中间宿主中传播的主要原因。细胞外囊泡(EV)可以将miRNA转移到寄生虫细胞中以调节mRNA表达。然而,加载发育途径相关的miRNA,例如与电动汽车中的Notch信号通路相关的信号通路尚不清楚。因此,我们在体外筛选了参与颗粒大肠杆菌包封过程中Notch途径的miRNA-mRNA亚网络,并评估了寄生虫和EV中表达的变化.
方法:使用高通量测序筛选PSC和微囊藻(MC)之间差异表达(DE)的mRNA和miRNA。将从转录组分析获得的DEmRNA与预测为小RNA文库的保守DEmiRNA的靶标的mRNA相交。使用公共数据库分析DEmiRNA功能,并建立了与Notch通路相关的miRNA-mRNA亚网络。验证了蠕虫和EV在不同时间的Notch通路相关mRNA和miRNA表达。
结果:总计,在来自转录组的1586个DEmRNA与使用来自小RNA文库的39个DEmiRNA预测的9439个靶mRNA之间的交叉之后,筛选MCs和PSC之间的1445个DEmRNA。DEmRNA聚集到94个代谢途径中,包括Notch通路.五个DEmiRNA,包括表达最显著的新DEmiRNA,egr-new-mir0694-3p,对应于四个靶mRNA(EgrG_000892700、EgrG_001029400、EgrG_001081400和EgrG_000465800)都富集在Notch途径中。上述mRNA和miRNA的表达与高通量测序结果一致,并验证各miRNA在EV中的表达。注释为Notch通路中的ADAM17/TACE,EgrG_000892700在PSC包封期间下调。egr-miR-4989-3p和egr-miR-277a-3p在包封后电动汽车中的表达是包封前电动汽车中的近五倍,这可能会调节EgrG_000892700的表达。
结论:对应于4个靶mRNA的5个miRNAs可能在PSC包封过程中参与Notch通路的调控。EV可能由于egr-miR-4989-3p和egr-miR-277a-3p的持续靶向而调控PSC中EgrG_000892700的表达,并参与Notch通路的调控。该研究可能会扩展通过EVmiRNAs阻断细粒大肠杆菌PSC继发感染的新思路。
BACKGROUND: Encystation of the protoscoleces (PSCs) of Echinococcus granulosus is the main cause of secondary hydatid dissemination in the intermediate host. Extracellular vesicles (EVs) can transfer miRNAs into parasite cells to regulate mRNA expression. However, loading of developmental pathway-related miRNAs, such as those related to the Notch signalling pathway in EVs is unclear. Thus, we screened the miRNA-mRNA subnetwork involved in the Notch pathway during E. granulosus encystation in vitro and assessed changes in expression in the parasite and EVs.
METHODS: mRNAs and miRNAs differentially expressed (DE) between PSCs and microcysts (MCs) were screened using high-throughput sequencing. DE mRNAs obtained from transcriptome analysis were intersected with mRNAs predicted to be targets of the conserved DE miRNAs of a small RNA library. DE miRNA functions were analysed using public databases, and a miRNA-mRNA subnetwork related to the Notch pathway was established. Notch pathway-related mRNA and miRNA expression of worms and EVs at different times was verified.
RESULTS: In total, 1445 DE mRNAs between MCs and PSCs were screened after the intersection between 1586 DE mRNAs from the transcriptome and 9439 target mRNAs predicted using 39 DE miRNAs from the small RNA library. The DE mRNAs were clustered into 94 metabolic pathways, including the Notch pathway. Five DE miRNAs, including the most significantly expressed new DE miRNA, egr-new-mir0694-3p, corresponding to four target mRNAs (EgrG_000892700, EgrG_001029400, EgrG_001081400 and EgrG_000465800) were all enriched in the Notch pathway. The expression of the above mRNAs and miRNAs was consistent with the results of high-throughput sequencing, and the expression of each miRNA in EVs was verified. Annotated as ADAM17/TACE in the Notch pathway, EgrG_000892700 was down-regulated during PSC encystation. egr-miR-4989-3p and egr-miR-277a-3p expression in EVs after encystation was nearly five times that in EVs before encystation, which might regulate the expression of EgrG_000892700.
CONCLUSIONS: Five miRNAs corresponding to four target mRNAs may be involved in regulating the Notch pathway during the PSC encystation. EVs may regulate the expression of EgrG_000892700 in PSCs because of continuous targeting of egr-miR-4989-3p and egr-miR-277a-3p and participate in the regulation the Notch pathway. The study might expand new ideas for blocking the secondary infection of E. granulosus PSCs via EVs miRNAs.