鳗弧菌感染下大菱鲆肠道黏膜免疫关键长链非编码RNA的鉴定、表达分析与功能研究

The mucosal immune system serves as the first line of host defense against pathogen infection, especially for the fishes, which are living in the pathogen-enriched aquatic environment. The mucosal surfaces of fish (skin, gill and intestine) are exposed to a wide range of pathogens, and the immune events are happening all the time to prevent the pathogen attachment and invasion on these surfaces. Although the long non-coding RNAs (lncRNAs) have been widely reported to be involved in the regulation of mucosal immune processes, the regulation mechanisms of lncRNAs in teleost intestinal mucosal immune processes are still poorly understood. To provide a comprehensive picture of the regulatory roles of lncRNAs in these processes, we aim to conduct a series of bioinformatics analysis to characterize one or two candidate key lncRNAs from the turbot intestine tissues during the infection of Vibrio anguillarum, using high-throughput sequencing datasets (including lncRNAs, microRNAs and mRNAs) from our research group. Moreover, we intend to characterize the expression signatures of those key lncRNAs not only among healthy turbot tissues but also during the infection processes of Vibrio anguillarum. In addition, we aim to build lncRNA-microRNA-mRNA coexpression networks to reveal the potential vital roles of those lncRNAs in immune-related pathways. Last but not the least, we aim to explore the functions of those key lncRNAs not only through the knockdown analysis of lncRNAs and their target genes but also through the analysis of the interactions between the lncRNAs and proteins. This proposal would set valuable foundations for future studies on the teleost mucosal immune regulatory mechanisms.

鱼类生存在病原体富集的水环境中，其黏膜表面屏障（皮肤、鳃和肠道）是机体抵抗病原体入侵的首要防线，而其中肠道黏膜免疫屏障在抵抗细菌入侵过程中发挥着至关重要的作用。长链非编码RNA（lncRNA）已被证实广泛参与黏膜免疫调控过程，然而关于lncRNA对于鱼类肠道黏膜免疫调节机制的研究还非常欠缺。因此，本项目拟通过生物信息学的方法分析已经完成高通量测序的鳗弧菌侵染过程中大菱鲆肠道粘膜lncRNA、mRNA及microRNA数据库，筛选1-2个候选关键lncRNA，并分析其在大菱鲆组织间分布及感染后多组织表达分布规律；通过构建lncRNA-microRNA-mRNA共表达网络，进行lncRNA及其靶基因功能缺失分析以及lncRNA与蛋白质互作分析，明确关键lncRNA对大菱鲆肠道黏膜免疫的调控机制。本项目的实施将为更好的研究硬骨鱼黏膜免疫调控机理提供宝贵的基础。

鱼类生存在病原体富集的水环境中，其黏膜表面屏障（皮肤、鳃和肠道）是机体抵抗病原体入侵的首要防线，而其中肠道黏膜免疫屏障在抵抗细菌入侵过程中发挥着至关重要的作用。长链非编码RNA（lncRNA）已被证实广泛参与黏膜免疫调控过程，然而关于lncRNA对于鱼类肠道黏膜免疫调节机制的研究还非常欠缺。因此，本项目拟通过生物信息学的方法分析已经完成高通量测序的鳗弧菌侵染过程中大菱鲆肠道粘膜lncRNA、mRNA及microRNA数据库，筛选1-2个候选关键lncRNA，并分析其在大菱鲆组织间分布及感染后多组织表达分布规律；通过构建lncRNA-microRNA-mRNA共表达网络，进行lncRNA及其靶基因功能缺失分析以及lncRNA与蛋白质互作分析，明确关键lncRNA对大菱鲆肠道黏膜免疫的调控机制。根据鳗弧菌分布结果，完成对大菱鲆黏膜免疫屏障在鳗弧菌感染前后（2h，12h和48h）的差异表达研究，共有158个lncRNA、50个circRNA和302个mRNA在3个时间点表现出不同的表达水平。构建了大菱鲆肠道黏膜免疫lncRNA-microRNA-circ-mRNA共表达网络，并筛选出两条关键的lncRNA：SETD3-OT和NLRP14-OT进行后续包括详细鉴定、敲降功能研究及蛋白互作等研究。鳗弧菌感染后，SETD3-OT在肠道的6h和12h出现显著下调。NLRP14-OT基因干扰效率达60%以上，且干扰后NLRP14-OT显著下调。相关研究结果已发表论文2篇，培养研究生3名，参加学术会议3次，项目负责人于2019年获批省级人才称号。结题一年内还将继续发表论文2-3篇。本项目研究结果丰富了鱼类黏膜免疫研究内容，为进一步深入探索大菱鲆弧菌的免疫防治和硬骨鱼黏膜免疫调控机制提供坚实的基础。