College of Animal Science and Technology, Yangzhou University, Yangzhou 225000, China.
Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
Int J Mol Sci. 2022 Feb 7;23(3):1875. doi: 10.3390/ijms23031875.
H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.
H1N1 和 H3N2 是两种最常见的猪流感病毒（SIV）亚型。它们不仅危及猪业，而且还存在巨大的人畜共患病风险。然而，猪（宿主）对抗流感病毒感染的分子机制和调控网络仍不清楚。在本研究中，构建了猪肺泡巨噬细胞（3D4/21）模型，感染猪流感病毒（H1N1 和 H3N2）。本研究揭示了 H1N1 和 H3N2 感染 3D4/21 细胞后 miRNA、mRNA、lncRNA 和 circRNA 的表达谱。然后，通过生物信息学分析、基因过表达、基因干扰、实时定量 PCR（qPCR）、双荧光素酶活性测定和 RNA 免疫沉淀（RIP）验证了两种 ceRNA（TCONS_00166432-miR10391-MAN2A1 和 novel_circ_0004733-miR10391-MAN2A1），这些 ceRNA 调控 3D4/21 细胞中 H1N1 和 H3N2 的感染。此外，通过 qPCR 和酶联免疫吸附试验（ELISA）鉴定了重要候选分子（miR-10391、TCONS_00166432 和 novel_circ_0004733）。最后，通过基因干扰、qPCR、Western blot 和 ELISA 鉴定了靶基因的调控作用及其可能的分子机制。本研究结果表明，TCONS_00166432 和 novel_circ_0004733 可以竞争性结合 miR-10391 来靶向基因，从而调节猪流感病毒感染 3D4/21 细胞。本研究首次报道了参与调节猪流感病毒感染 3D4/21 细胞的 ceRNA 网络，为 3D4/21 细胞对抗猪流感病毒感染的分子机制提供了新的见解。
