PCV2感染猪肺泡巨噬细胞自噬过程中miRNA差异表达谱及靶基因功能调控网络研究

Porcine alveolar macrophages （PAM） is not only the target cells of PCV2 but also an important immune cells. PCV2 could infect PAM, not result in apoptosis, escape the cellular immune identify and cause immunosuppression. Autophagy can inhibit apoptosis and virus can escape cell-mediated immunity by miRNA interfere with the normal immune process of host cells. We assumed that PCV2 infection may lead to changes of miRNA regulation and autophagy of alveolar macrophages, thereby inhibiting apoptosis, evading the immune system and causing immunosuppression. The results of our preliminary study indicated that PCV2 infection could cause autophagy on the alveolar macrophages. Therefore, we use the porcine alveolar macrophage cell lines as a model to explore the relationship between PCV2, autophagy and PAM. miRNA microarray is used to detect differential expression of miRNA in the autophage process of PCV2-infected PAM. The way of biological information is used to predict the target gene. And the target gene is tested and verified, then construct the regulatory networks between the specific expression of microRNA and the target gene to get the critical miRNA which regulated replication of PCV2 on PAM. The interaction between the miRNA and target gene is validated to analyze the impact of miRNA on the biological function of the target gene. The main specific expression of miRNA, the target genes and the gene fuction which regulated by specific miRNA will reveal that the miRNA whether inhibit the apotosis of PAM and the mechanism of immune evasion of PCV2. These results of this study have a certain theoretical significance and application value.

猪肺泡巨噬细胞（PAM）不仅是PCV2的靶细胞,而且是重要的免疫细胞。PCV2可以感染PAM、不引起凋亡，逃避细胞免疫识别引起免疫抑制，而自噬可抑制凋亡产生。病毒可通过miRNA干扰宿主细胞的正常免疫过程，实现免疫逃逸。因而，我们设想PCV2感染可能会引起miRNA调控的改变和巨噬细胞发生自噬，从而抑制凋亡产生，逃避免疫，引起免疫抑制。本课题前期研究表明，PCV2感染PAM可引起自噬现象。因此，拟以猪肺泡巨噬细胞系为模型，探讨PCV2-自噬-PAM之间的关系；对自噬过程中的PAM进行miRNA差异表达分析；预测差异表达miRNA的靶基因并进行验证，绘制靶基因调控网络，确立调控病毒在肺泡巨噬细胞中复制和自噬的miRNA；通过miRNA与靶基因mRNA相互作用的功能学验证，确立miRNA对靶基因生物学功能的影响；验证miRNA是否抑制PAM凋亡，探索PCV2免疫逃避机制，具有一定的理论意义和应用价值。

猪肺泡巨噬细胞（PAM）不仅是PCV2的靶细胞,而且是重要的免疫细胞。PCV2可以感染PAM、不引起凋亡，逃避细胞免疫识别引起免疫抑制，而自噬可抑制凋亡产生。病毒可通过miRNA干扰宿主细胞的正常免疫过程，实现免疫逃逸。因而，我们设想PCV2感染可能会引起miRNA调控的改变和巨噬细胞发生自噬，从而抑制凋亡产生，逃避免疫，引起免疫抑制。本课题前期研究表明，PCV2感染PAM可引起自噬现象。因此，拟以猪肺泡巨噬细胞系为模型，探讨PCV2-自噬-PAM之间的关系；对自噬过程中的PAM进行miRNA差异表达分析；预测差异表达miRNA的靶基因并进行验证，绘制靶基因调控网络，确立调控病毒在肺泡巨噬细胞中复制和自噬的miRNA；通过miRNA与靶基因mRNA相互作用的功能学验证，确立miRNA对靶基因生物学功能的影响；验证miRNA是否抑制PAM凋亡。结果表明，PCV2感染3D4/21细胞和自噬之间存在着正相关性，PCV2感染诱导的自噬促进了PCV2的感染。过表达miR-30a-5p显著促进PCV2的感染和自噬现象，且具有浓度依赖性。抑制miR-30a-5p的表达显著降低了PCV2的复制也验证了这一结果。进一步的研究表明miR-30a-5p具有调节PCV2感染和宿主免疫系统的作用。此外，miR-30a-5p并不直接靶向PCV2 基因组，而是靶向14-3-3基因，通过调节细胞周期影响PCV2的复制和自噬。这些发现不仅为研究PCV2 感染过程中病毒与宿主之间的关系提供了新的思路，也为防治PCV2感染提供了新的抗病毒策略。