Regulatory effect and mechanism of APN gene on porcine epidemic diarrhea virus resistance.
Regulatory effect and mechanism of APN gene on porcine epidemic diarrhea virus resistance.
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DOI：
10.1016/j.gene.2021.145448
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发表时间：
2021-01
期刊：
影响因子：
3.5
通讯作者：
Shiqin Wang;Chao Xu;Jing Shi;Haifei Wang;Shenglong Wu;W. Bao
中科院分区：
文献类型：
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作者：
Shiqin Wang;Chao Xu;Jing Shi;Haifei Wang;Shenglong Wu;W. Bao
目的猪流行性腹泻病毒（PEDV）传染性与猪氨基肽酶N （APN）表达水平有明显相关性。本研究旨在探讨apc表达水平在PEDV感染反应中的调节机制。方法采用实时荧光定量PCR检测PEDV感染后不同时间点的基因表达动态。随后，利用CRISPR/Cas9基因编辑技术，生成aapn敲除IPEC-J2细胞系，碘化丙烯染色研究apnon对细胞增殖的影响，间接免疫荧光法检测抗pedv活性。最后，通过亚硫酸酯测序PCR和双荧光素酶报告基因测定分析了apnpromoter区域单核苷酸多态性（snp）和甲基化对基因表达的影响。结果感染后4 ~ 24 h， ap1表达显著上调。apn敲除后IPEC-J2细胞株的细胞活性在不同时间点均受到明显抑制。此外，细胞周期分析表明g1期细胞数量增加，s期细胞数量显著减少。此外，调节G1期的关键周期因子在apn敲除细胞中高度表达。apn敲除细胞感染PEDV后24 h内，病毒颗粒RNA拷贝数、抗病毒基因和炎症因子mRNA水平均显著降低。同样，间接免疫荧光法证实PEDV颗粒数量明显减少。序列分析显示apnpromoter区域存在两个CpG岛。然而，apnpromoter的甲基化状态与mRNA水平之间没有明显的相关性。双荧光素酶报告基因检测显示，rs326030589 (G/A) SNP显著提高了apn的启动子活性。结论apn基因敲除可增强IPEC-J2细胞对PEDV的抗性。此外，apnpromoter区域的rs326030589参与基因转录调控。本研究结果为研究apnn基因的调控机制提供了参考，并可能为apnn基因在猪流行性腹泻耐药育种中的应用提供参考。
PurposeThe expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulatingAPNexpression level in response to PEDV infection.MethodsQuantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate aAPN-knockout IPEC-J2 cell line, exploring the effects ofAPNon cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in theAPNpromoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay.ResultsAPNexpression was significantly upregulated within 4–24 h post-infection. The cytoactivity of theAPN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed inAPN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24 h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in theAPNpromoter region. However, there was no evident correlation between the methylation status ofAPNpromoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity ofAPN.ConclusionsThese results suggested thatAPNknockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in theAPNpromoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulatingAPNand may contribute to the application ofAPNgene in resistance breeding of swine epidemic diarrhea.