日本血吸虫卵巢发育关键miRNAs调控虫体生殖发育和产卵的分子机理研究

Schistosomes are dioecious trematodes responsible for schistosomiasis in humans and other animals. Sexual maturation and subsequent egg production in female schistosomes require a permanent pairing with male schistosomes at the gynecophornal canal. Matured female schistosomes produce a large number of eggs which are primarily responsible for the pathogenesis of schistosomiasis as well as dissemination of the parasite. However, the current understanding of molecular mechanism of female sexual maturation and egg production is very limited. In our previous study, we identified 38 Schistosoma japonicum miRNAs, including 10 previously unknown miRNAs, using small RNA deep sequencing in the key stages of male-female pairing, gametogenesis, and egg production. We further identified 30 potential target genes for 16 of the S. japonicum miRNAs using antibody-based pull-down assays and bioinformatic analyses. We further validated that Smad1 is a target gene of Bantam miRNA and Frizzled7 is a target gene of miR-31 using either in vitro luciferase assays or in vivo miRNA suppression experiments. Notably, suppression of the female enriched miRNAs bantam and miR-31 led to morphological alteration of ovaries in female schistosomes. These findings suggest the key miRNAs (Bantam miRNA and miR-31) and their targets as well as the related signal pathways play an important role in the regulation of schistosome development, sexual maturation and egg production. Therefore, we propose to use RNA seq, quantitative proteomics and bioinformatics to systemically determine the target genes of the key miRNAs involved in ovarian development in S. japonicum. Next, we will use Argonaute antibody-based pull-down, in vitro luciferase assay and in vivo miRNA suppression assay to validate the target genes of the key miRNAs. Then, we will also investigate the partners of miRNA targets and their signal pathways involved in schistosome development and sexual maturation by co-immunoprecipitation and yeast two hybrid system. Finally, we plan to apply miRNA inhibitor, RNA interference or chemical inhibitors to block the functions of several key molecules and/or their involved signal pathways to evaluate the potential of these molecules as drug targets against schistosomiasis. The expected results of the proposal will not only provide important information for theoretically understanding the mechanism of schistosome development and sexual maturation but also may facilitate to develop effective anti-schistosome vaccines and drugs for schistosomiasis control.

血吸虫雌雄异体，雌雄虫合抱是雌虫性发育成熟的前提，而性发育成熟的雌虫所产的虫卵又是造成宿主病理损害和疾病传播的根本原因。目前，有关血吸虫生殖发育的分子机理认识仍非常有限。我们发现血吸虫雌虫高表达Bantam miRNA和miR-31具有调控卵巢发育的重要功能，并表明Bantam miRNA和miR-31可分别靶向作用TGF-Beta和WNT信号通路中Smad1和Frizzled7分子。为此，本项目拟系统地研究发现血吸虫雌虫卵巢发育关键miRNAs（Bantam miRNA和miR-31）的靶向作用分子，揭示关键miRNAs通过与重要靶向分子的互作所调控的信号转导通路，探明关键miRNAs及其调控的信号通路中的重要分子在血吸虫性成熟和产卵中的功能，为深入阐明血吸虫性成熟和产卵的分子机制奠定理论基础。研究结果不仅对血吸虫生殖发育机理的研究具有理论意义，还对血吸虫病的防治实践具有重要现实意义。

血吸虫雌雄异体，雌雄虫合抱是雌虫性发育成熟的前提，而性发育成熟的雌虫所产的虫卵又是造成宿主病理损害和疾病传播的根本原因。目前，有关血吸虫生殖发育的分子机理认识仍非常有限。我们前期研究发现血吸虫雌虫高表达Bantam miRNA和miR-31具有调控卵巢发育的重要功能。本项目系统地研究血吸虫雌虫卵巢发育关键miRNAs（Bantam miRNA和miR-31）的靶向作用分子及重要分子参与血吸虫生殖发育和产卵的调控功能。具体发现为：1）揭示了抑制关键miRNAs（Bantam miRNA 和miR-31）导致卵巢发育异常的分子基础；2) 发现了Bantam miRNA调控Sec-Rho-Rad信号通路，进而参与虫体卵巢的发育调控；3）明确了Bantam/miR-31潜在调控Hippo信号通路中的Friz5/7/9基因，并影响血吸虫的寄生和产卵的新功能，且抑制血吸虫Hippo信号通路中的Friz7信号分子可显著影响血吸虫的寄生和产卵。另外，本项目还发现了血吸虫Bantam miRNA潜在调控虫体Long non-coding RNAs的表达；血吸虫外泌体相关miR-750调控雌虫卵巢发育的功能。总之，本项目研究揭示了血吸虫发育关键miRNAs（Bantam miRNA和miR-31）的靶向作用分子及重要靶向分子的互作分子所调控的信号转导通路及该通路在血吸虫性成熟和产卵中的功能。上述研究发现为深入阐明血吸虫性成熟和产卵的分子机制奠定了理论基础。研究结果不仅对深入理解血吸虫生殖发育的机理具有理论意义，还对血吸虫病的防治实践具有重要现实意义。