日本血吸虫性别差异表达MicroRNAs功能研究

Schistosomiasis japonica, one of the most serious parasitic diseases, remains a major public health concern in China. Its causative pathogen, Schistosoma japonicum has a complex life cycle and gene regulation patterns. Exploring the gene regulation will help us to learn more about biological mechanism of schistosome. Our previous studies have revealed that, as an important regulatory factor, microRNAs (miRNAs) in S. japonicum are expressed in a stage- and gender-biased manner. However, the biological functions of these miRNAs still have been less explored. The project here aims to define tissue localization of sex-differentially expressed miRNAs by ISH in separated adult worms, to predict the target genes of sex-differentially expressed miRNAs by bioinformatic methods and verify the miRNA-target gene interactions by experimental approaches. We will validate these interactions by miRNAs overexpression or knockdown in vivo, and further reveal the potential roles of sex-differentially expressed miRNAs based on the functional hints of target genes and/or phenotype changes after target gene manipulation. This research will help to elucidate the of gene regulatory functions of sex-differentially expressed miRNAs in S. japonicum during the adaptation of the dioecious lifestyle, the maintenance of sex discrimination, and in the context of parasite-host interplay, and will provide novel theoretical basis for Schistosomiasis control.

日本血吸虫病是我国危害最为严重的寄生虫病，也是重要的公共卫生问题之一。日本血吸虫具有复杂的生活史和基因调控模式，对基因表达调控的研究将有助于了解血吸虫的生物学规律。本课题组前期研究表明，作为基因表达调控的重要因子，日本血吸虫miRNA呈现期偏好性表达和性别差异表达，但是这些miRNAs的生物学功能尚不清楚。本项目拟明确日本血吸虫性别差异表达miRNAs在成虫中组织定位；利用生物信息学手段预测并实验验证性别差异表达miRNA的靶基因；通过虫体miRNA的过表达或knockdown进一步体内验证性别差异表达miRNA的靶基因；根据靶基因的功能提示和/或靶基因变化引起的虫体表型改变推断性别差异表达miRNA的潜在功能。本研究将有助于阐明日本血吸虫性别差异表达miRNAs在适应雌雄异体生活方式，维持性别分化状态，及与宿主相互作用过程中的基因表达调控功能，为血吸虫病的防控提供新的理论依据。

血吸虫病是全球最为严重的公共卫生问题之一，也是我国最为严重的寄生虫病。血吸虫生活史复杂，防控难度大。血吸虫具有基因表达调控模式，对基因表达调控的研究将有助于了解血吸虫的两性及发育生物学。本课题组前期研究表明，作为基因表达调控的重要因子，日本血吸虫miRNAs呈现期偏好性表达和性别差异表达，但是这些miRNAs的基因调控尚不清楚。本项目构建不同发育期非编码小RNA文库，构建日本血吸虫miRNA分期转录表达谱；并实验验证不同发育阶段miRNA表达。设计构建到目前为止覆盖最全面的第二代日本血吸虫全基因组基因芯片（含有41,982个探针），利用此芯片深度分析日本血吸虫雌雄成虫及不同发育时期富集表达mRNA转录本。分析雌雄差异表达基因发现约为1/10的血吸虫基因呈现性别偏好的表达，其中685个基因在雄虫中高表达，430个基因则在雌虫中高表达。分析期富集表达发现328，95，268和532个基因分别在尾蚴，肝期童虫，成虫和虫卵中高表达。生物信息学针对上述mRNA转录本预测miRNAs靶基因，总体上看，约一半的基因可受到miRNAs的调节。其中约68%的位点位于mRNA的CDS区，约23%的位点位于mRNA的3’ UTR区，约9%的位点位于mRNA的5’ UTR区。其中7个miRNAs (sja-let-7, sja-miR-1, sja-miR-71, sja-miR-71b-5p, sja-miR-7-5p, sja-miR-190-5p和sja-miR-3479-5p) 具有丰富的靶位点。 本研究有助于阐明日本血吸虫miRNA在适应雌雄异体生活方式，维持不同发育状态，及与宿主相互作用过程中的基因表达调控功能，为血吸虫病的防控提供新的理论依据。