外泌体miRNA在拟穴青蟹抗病毒免疫调控中的功能研究

It is reported that exosome plays an essential role during the host-virus interaction, while the packaged microRNA (miRNA) is critical for its functioning. Scylla paramamosain is an ideal carrier for exosome to function since it possesses open circulatory system, but few reports on the area. In the previous study, for the first time, we isolated exosomes with high-purity from the hemolymph of Scylla paramamosain, the follow-up study showed that the exosomes isolated from WSSV-infected Scylla paramamosain could efficiently inhibit virus replication, indicating that exosome may participate in the anti-viral immunoregulation of Scylla paramamosain. Based on the existing study, we intend to take miRNA as the breakthrough point，through performing miRNA sequencing of the exosomes isolated from WSSV-infected/non-infected Scylla paramamosain, miRNAs abnormally regulated in the exosomes will be identified, the prediction and validation of the target genes will also be conducted, finally, the function and molecular mechanism of exosomal miRNA during the anti-viral immunoregulation of Scylla paramamosain will be revealed through the screening of the target gene-interaction proteins and the exploration of downstream signaling pathways. The relevant research findings are expected to obtain novel insight of the immune mechanism of Scylla paramamosain, and have a positive guiding significance for disease prevention and control of Scylla paramamosain aquaculture.

研究表明外泌体在宿主-病毒互作过程中扮演着关键的角色，其包裹的微小RNA（miRNA）对其功能的发挥至关重要。拟穴青蟹具备开放式体液循环系统，是外泌体免疫途径行使功能的理想载体，但却鲜有相关的报道。在课题组的前期研究中，我们首次从拟穴青蟹血淋巴中分离到高纯度的外泌体，并发现拟穴青蟹被白斑综合症病毒（WSSV）感染后其血淋巴分泌的外泌体能显著抑制病毒的复制，预示着外泌体可能参与了拟穴青蟹的抗病毒免疫调控。本项目拟在已有研究的基础上，以miRNA为切入点，通过对WSSV感染前后拟穴青蟹的外泌体进行miRNA测序，进而明确外泌体中异常调控的miRNA，并对其靶基因进行预测及验证，最后通过靶基因互作蛋白的筛选及下游信号通路的检测来揭示外泌体miRNA在拟穴青蟹抗病毒免疫调控中的功能及分子机制。相关研究成果有望获得对拟穴青蟹免疫机制的新认识，在实践上对拟穴青蟹的病害防治具有积极的指导意义。

许多研究表明外泌体是一种宿主抵御病毒侵染的重要防卫工具，在宿主细胞-病毒互作过程中扮演着关键的角色，但低等动物尤其是甲壳动物中却鲜有相关方面的报道。项目组在前期研究中首次从拟穴青蟹血淋巴中分离到高纯度的外泌体并进行了相关鉴定，发现被WSSV感染的拟穴青蟹血淋巴中所分泌的外泌体能有效抑制病毒的复制。本项目拟在此基础上，以外泌体所包裹的miRNAs为切入点，通过对WSSV感染前后拟穴青蟹血淋巴中的外泌体进行miRNA测序，明确期间异常调控的miRNA并对其靶基因及相关功能进行分析，进而揭示外泌体miRNA在拟穴青蟹抗病毒免疫调控中的功能及其分子机制。研究结果表明病毒感染过程中，青蟹血淋巴外泌体所包裹miR-137和miR-7847含量减少，而miR-137和miR-7847均可靶向凋亡诱导因子AIF，导致青蟹体内AIF激活。AIF一方面入核促进基因组DNA的降解，另一方面可以同凋亡蛋白Bax 竞争性结合HSP70，导致Bax-HSP70复合体的瓦解，而游离的Bax进一步转位至线粒体激活线粒体凋亡程序并以此来抵御病毒的侵染。上述成果揭示了外泌体miRNAs介导拟穴青蟹抗病毒免疫的调控通路，发现了甲壳动物清除体内病原微生物的新机制，对于解决困扰青蟹养殖业的病害防治难题具有重要的实际意义。