乙型脑炎病毒感染PK15细胞的lncRNA差异表达谱分析

doi:10.11843/j.issn.0366-6964.2022.01.027

摘要/Abstract

摘要： 旨在分析乙型脑炎病毒（Japanese encephalitis virus，JEV）感染猪肾上皮细胞PK15后的lncRNA差异表达谱，探究宿主lncRNA在JEV感染和宿主防御中的潜在功能。本研究中利用JEV感染PK15细胞，感染36 h，收集细胞，同时设立未感染对照组，每组3个生物学重复。提取细胞总RNA，构建cDNA文库后进行高通量测序，筛选出差异表达lncRNA。通过对差异表达lncRNA与mRNA的位置关系以及序列相似性预测顺式和反式作用的靶基因，将预测的靶基因进行GO和KEGG分析。最后随机选出9个差异表达lncRNAs，利用实时荧光定量PCR （quantitative reverse transcription PCR，qRT-PCR）技术进行表达验证。结果表明，本研究中共鉴定出856个差异表达lncRNAs，其中452个lncRNAs显著上调表达，404个lncRNAs显著下调表达。GO分析发现，lncRNA的靶基因主要富集于先天性免疫反应；KEGG通路分析发现lncRNA的靶基因显著富集于肿瘤坏死因子、Toll样受体和NF-κB等信号通路。通过qRT-PCR验证发现，定量结果和测序结果基本一致。本研究为进一步探究lncRNA参与宿主免疫反应调控JEV复制奠定了理论基础。

关键词: 猪, 乙型脑炎病毒, 先天免疫反应, PK15细胞, 高通量测序

Abstract: The objective of this study was to analyze the differential expression profile of lncRNA in pig kidney epithelial cells PK15 infected by Japanese encephalitis virus (JEV), and to explore the potential function of host lncRNA in JEV infection. In this study, JEV was used to infect PK15 cells for 36 hours, and the cells were collected. Meanwhile, an uninfected control group was established. There were 3 biological replicates in each group. The total RNA was extracted, and the cDNA libraries were constructed and then subjected to high-throughput sequencing to identify differentially expressed lncRNA. The cis- and trans-acting target genes of lncRNA were obtained by calculating the positional relationship and sequence similarity between differentially expressed lncRNA and mRNA. Next, the predicted target genes were analyzed by GO and KEGG. Finally, 9 differentially expressed lncRNAs were randomly selected for expression verification using real-time fluorescent quantitative PCR (quantitative reverse transcription PCR, qRT-PCR) technology. The results showed that a total of 856 differentially expressed lncRNAs were identified, of which 452 lncRNAs were significantly up-regulated and 404 lncRNAs were significantly down-regulated. GO analysis results showed that the target genes of lncRNAs were mainly enriched in innate immune response; KEGG pathway analysis results showed that the target genes of lncRNA were significantly enriched in signal pathways such as tumor necrosis factor, Toll-like receptor and NF-κB. The quantitative results are the same as the sequencing results through qRT-PCR verification. This study lays a theoretical foundation for further exploring the roles of lncRNA in JEV infection.

Key words: swine, Japanese encephalitis virus, innate immune response, PK15 cells, high-throughput sequencing

中图分类号:

S855.3

朱静静, 戴政列, 汪涵, 李向臣, 赵阿勇, 周晓龙, 杨松柏. 乙型脑炎病毒感染PK15细胞的lncRNA差异表达谱分析[J]. 畜牧兽医学报, 2022, 53(1): 272-281.

ZHU Jingjing, DAI Zhenglie, WANG Han, LI Xiangchen, ZHAO Ayong, ZHOU Xiaolong, YANG Songbai. Analysis of Differential Expression Profile of LncRNA in PK15 Cells Infected with Japanese Encephalitis Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 272-281.

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