Key Laboratory of Tropical Diseases and Control, Ministry of Education, Guangzhou, People's Republic of China.
Parasitol Res. 2014 May;113(5):1883-96. doi: 10.1007/s00436-014-3836-6. Epub 2014 Apr 3.
The pathogenesis of angiostrongyliasis, resulting from the third-stage and the fourth-stage Angiostrongylus cantonensis larvae invasion of the human central nervous system, remains elusive. MicroRNAs are important regulators of gene expression and involved in many biological processes. The aim of this study was to determine and characterize miRNAs of third (L3) and fourth (L4) larvae of A. cantonensis by Solex deep sequencing. A total of 629 conserved miRNAs (526 and 376 miRNAs in L3 and L4 larvae of A. cantonensis, respectively) and three novel candidate miRNA from L3 and L4 larva of A. cantonensis were identified with bioinformatic analysis. There were 163 miRNAs upregulated and 54 miRNAs downregulated (fold changes ≥5.0) in the L4 of A. cantonensis compared with that of L3 of A. cantonensis. Interestingly, Gene Ontology "biological process" classifications revealed that 26 miRNAs of significantly differential expression are associated with the immune system, which implies that these miRNAs might participate in the pathogenesis of angiostrongyliasis by regulating genes involved in immune response pathways. Furthermore, the differential expression patterns of 26 conserved miRNAs between L3 and L4 of A. cantonensis were verified. The results of real-time PCR and Northern blot showed that the aca-miR-124 and aca-miR-146a-5p have a low level expression in L3 larvae but high level expression in L4 larvae. Transfection of aca-miR-124 mimics alone significantly downregulated the mRNA expression of IL-6 and IL-1β and TNF-a in the N9 cells, compared to the combination transfection of aca-miR-124 mimics and inhibitor (P < 0.05), suggesting that miR-124 of A. cantonensis have an important role in the suppression of microglia activation. In conclusion, the study presents a general picture of the expression of small RNAs in L3 and L4 of A. cantonensis and highlights conserved miRNAs differentially expressed between L3 and L4 larvae. Our data revealed that miRNAs of parasite may mediate important roles in A. cantonensis immune evasion and aca-miR-146a-5p can serve as a potential biomarker to diagnose angiostrongyliasis.
旋毛虫病的发病机制是由第三期和第四期广州管圆线虫幼虫侵入人体中枢神经系统引起的，但仍不清楚其发病机制。microRNAs 是基因表达的重要调节因子，参与许多生物过程。本研究旨在通过 Solex 深度测序确定和描述广州管圆线虫第三期（L3）和第四期（L4）幼虫的 microRNAs。通过生物信息学分析，在 L3 和 L4 幼虫中分别鉴定出 526 个和 376 个保守 microRNAs（L3 和 L4 幼虫的 629 个保守 microRNAs 和 3 个新型候选 microRNA）。与 L3 相比，L4 中 163 个 microRNAs 上调，54 个 microRNAs 下调（倍数变化≥5.0）。有趣的是，GO“生物过程”分类显示，26 个差异表达的 microRNAs 与免疫系统有关，这意味着这些 microRNAs 可能通过调节参与免疫反应途径的基因参与旋毛虫病的发病机制。此外，还验证了 L3 和 L4 之间 26 个保守 microRNAs 的差异表达模式。实时 PCR 和 Northern blot 结果表明，aca-miR-124 和 aca-miR-146a-5p 在 L3 幼虫中表达水平较低，而在 L4 幼虫中表达水平较高。与 aca-miR-124 模拟物和抑制剂的组合转染相比，单独转染 aca-miR-124 模拟物可显著下调 N9 细胞中 IL-6、IL-1β 和 TNF-a 的 mRNA 表达（P<0.05），表明旋毛虫的 miR-124 在抑制小胶质细胞激活方面具有重要作用。总之，本研究描绘了 L3 和 L4 广州管圆线虫中小 RNA 的表达图谱，并强调了 L3 和 L4 幼虫之间差异表达的保守 microRNAs。我们的数据表明，寄生虫的 microRNAs 可能在广州管圆线虫的免疫逃避中发挥重要作用，aca-miR-146a-5p 可以作为诊断旋毛虫病的潜在生物标志物。
