cfa-miR-143在调控犬流感病毒致病机制的研究

Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory disease in dogs. Recently, numerous cases have been reported that CIV can break host barrier and infect cats, threatening human health. Therefore, it is very important to investigate the underlying mechanism of its infection and transmission. MicroRNAs can regulate gene expression by inhibition of mRNA translation and degradation of mRNAs via complementary binding with target sequences. It has been demonstrated that microRNAs play an important role in a variety of metabolic pathways. Interesting results have been observed in our previous studies, that microRNAs regulated the metabolic pathways. Thus, screening of differentially expressed microRNAs will be conducted in the following research. Cfa-miR-143 was found to be one of the differentially expressed microRNAs, via sequencing and analysis using bioinformatic software. For further study, we will focus on prediction of its target genes by biological software, and reference to relevant research data with signaling pathways relating to pathogenicity. The discovery of related target genes and signaling pathways would shed lights on invention of novel drugs or DNA vaccine. This would ultimately benefit for effectively canine influenza prevention and control. The completion of this study would also provide basic theoretical information for further understanding of molecular mechanisms in pathogenicity and dissemination of CIV.

犬流感病毒(CIV)是引起犬的急性呼吸道疾病的病原。近几年，世界多地先后报道流感病毒能突破宿主障碍感染犬、猫，对人类健康造成威胁，因此，研究CIV的感染与传播机理具有十分重要的意义。microRNAs可与靶mRNA分子互补结合抑制蛋白翻译或导致其降解，从而调控靶基因表达。已被证实在多种代谢途径中发挥重要作用。本课题组目前在研的项目中发现，多个microRNA在代谢途径起重要调控作用。所以，拟在上一个基金基础上，进行差异表达的microRNAs筛选，选取差异性表达最大的microRNA（即：cfa-miR-143）进行研究，通过生物学软件进行靶标预测，并参考相关研究资料挑选与致病性和靶标共相关的信号通路进行深入探索。通过相关靶标和通路探索性研究，将有助于发现新的药物作用靶标或设计新的核酸疫苗，为犬流感的有效防治和深入研究提供新的参考。同时对阐明犬流感病毒致病与传播的分子机制提供重要理论依据。

宿主microRNA能调节机体做出抗病毒感染的应答，主要包括免疫调控、靶向病毒基因。基于microRNA研究背景，通过荧光定量PCR、双荧光素酶检测、Western blot、间接免疫荧光和流式细胞术等实验技术，我们探索了cfa-miR-143在MDCK细胞中对抗犬流感感染的机制。本研究证实在MDCK细胞感染犬流感病毒的早期检测到cfa-miR-143上调表达，通过生物信息学软件预测cfa-miR-143的靶基因，我们选定与细胞生长、分化、凋亡有密切关系的靶基因，胰岛素样生长因子结合蛋白5基因(Igfbp5)。双荧光报告载体检测体统和cfa-miR-143的过表达实验，验证了cfa-miR-143能与Igfbp5的3’端非编码区进行结合，并负调控Igfbp5的表达。为了理解cfa-miR-143在CIV感染细胞中的作用，通过microRNA 激动剂和抑制剂，同时配合小RNA干扰实验，进一步研究基于目标基因的cfa-miR-143与病毒诱导细胞凋亡通路的关系。研究首先证实了，CIV感染后，MDCK细胞激活了凋亡通路对抗病毒感染，过表达cfa-miR-143显著提高了宿主的这一功能，增加了凋亡细胞数量，减少了病毒在胞内的增殖和细胞外的分泌量。随后，MDCK细胞预先共转染cfa-miR-143激动剂和TP53或者caspase3的抑制剂后，进行CIV感染实验。蛋白免疫印迹、流式细胞术、荧光定量PCR、和TCID50检测结果揭示，上调cfa-miR-143水平，可以活化p53-Caspase3凋亡信号通路，显著增加凋亡数量，抑制病毒的复制。因此，我们提出了cfa-miR-143在抗H3N2 CIV病毒感染中起到了积极的作用。