猪伪狂犬病毒Fa株侵染对PK-15细胞microRNAs表达谱的影响

Differential expression of microRNAs in PK-15 cells infected with Pseudorabies virus-Fa strain

摘要:

【目的】分析猪伪狂犬病毒Fa株(PRV-Fa)侵染对猪肾传代细胞PK-15 microRNAs (miRNAs)表达谱的影响。【方法】利用Illumina高通量测序技术，鉴定感染和非感染PRV-Fa的PK-15细胞的miRNAs；筛选并利用实时荧光定量RT-PCR (RT-qPCR)验证差异表达miRNAs；对差异miRNAs进行靶基因预测和Gene ontology (GO)分析。【结果】在感染和未感染PK-15细胞中分别检测到384个和405个miRNAs，其中感染PRV-Fa后差异表达的miRNAs共127个(60个上调，67个下调)。荧光定量结果显示差异miRNAs的表达趋势与高通量测序结果一致。GO分析显示，miRNAs广泛参与信号传导、细胞代谢、免疫反应、基因表达等生物学进程，其中miR-10b、miR-16、miR-18a、miR-19b、miR-20a、miR-145-5p、miR-146a、miR-181a、miR-499-5p等miRNAs与免疫相关。在靶基因调控网络图中，ssc-miR-30a-5p与ssc-miR-30d处于关键位置。研究鉴定出5个新的病毒编码miRNAs，其中PRV-miR-LLT2与PRV-miR-LLT4靶向PRV早期蛋白基因EPO。【结论】伪狂犬病毒Fa株感染对PK-15细胞编码miRNAs有显著影响。

Abstract:

[Objective] The aim was to analyze the microRNAs expression in PK-15 cells infected with Pseudorabies virus-Fa. [Methods] Illumina deep sequencing was used to identify and analyze the differentially expressed miRNAs in PK-15 cells with or without PRV-Fa infection. The sequencing results for selected miRNAs were confirmed with RT-qPCR. The host target genes were predicted and Gene Ontology (GO) analysis was done. [Results] We identified 384 and 405 miRNAs in the infected and uninfected cells, respectively. Overall, 127 miRNAs were expressed significantly differently after infected with PRV-Fa (60 upregulated and 67 downregulated). RT-qPCR showed that the expressions of miRNAs were consistent with the sequencing results. GO analysis showed that miRNAs were widely involved in signal transduction, cells metabolism, immune response, and gene expression. And miR-10b, miR-16, miR-18a, miR-19b, miR-20a, miR-145-5p, miR-146a, miR-181a and miR-499-5p were associated with immune response. In the regulatory network of target genes, ssc-miR-30d and ssc-miR-30a-5p were in the key positions. Moreover, five miRNAs encoded by PRV-Fa were identified, PRV-miR-LLT2 and PRV-miR-LLT4 were targeted to PRV EPO gene. [Conclusion] The infection of PRV-Fa has significant influence on PK-15 cells encoding miRNAs.