lncRNA在乙脑病毒感染PK15细胞过程中的作用研究

doi:10.16431/j.cnki.1671-7236.2019.07.020

摘要/Abstract

摘要：

本研究旨在初步探索乙脑病毒（JEV）感染PK15细胞后的增殖情况，筛选与病毒感染相关的lncRNA，并对其进行亚细胞定位及靶基因预测。通过免疫荧光试验来检测病毒结构蛋白E的表达情况，采用TCID₅₀法检测PK15细胞中病毒的增殖情况，利用实时荧光定量PCR检测病毒感染后lncRNA的表达水平，在NONCODE数据库对lncRNA进行亚细胞定位，通过starBase、NONCODE、KEGG等数据库对其进行靶基因预测和信号通路分析。结果显示，JEV感染PK15细胞后，24~36 h为病毒滴度指数增长期，感染后36 h病毒滴度已达10^-5.75 TCID₅₀/mL。PK15细胞在感染JEV 12 h后，lncRNA A、B、C表达水平均无显著变化（P>0.05），lncRNA D表达水平极显著下降（P<0.01）；感染JEV 24、36和48 h后lncRNA A、B、C表达水平极显著上升（P<0.01），lncRNA D表达水平极显著下降（P<0.01）。lncRNA A主要定位在胞质溶胶，lncRNA B在细胞核和细胞质中均有分布，lncRNA C主要定位在细胞质中，在细胞核中也有可能分布，lncRNA D可能在细胞内呈现广泛性分布。通过靶基因预测和信号通路分析，lncRNA A、B、C的靶基因主要为OAS1、OAS2、OASL、COX1等，lncRNA D的靶基因主要为DST、ND1、ND2、ND4等。信号通路分析发现lncRNA可能通过肿瘤坏死因子（TNF）、NF-κB和Toll样受体（TLR）等信号通路参与病毒感染后的增殖过程。本研究为进一步探索宿主细胞lncRNA对病毒增殖的影响奠定一定的基础。

关键词: lncRNA; PK15细胞; 乙脑病毒(JEV); 增殖

Abstract:

The aim of this study was to initially explore the proliferation of Japanese encephalitis virus (JEV) infected with PK15 cells,screen the lncRNAs associated with viral infections,and predict its subcellular localization and target genes.The expression of viral structural protein E was detected by immunofluorescence,the proliferation of virus in PK15 cells was detected by TCID₅₀ method,and Real-time PCR was used to detect the expression level of lncRNA after viral infection.Subcellular localization of lncRNA was carried out in the NONCODE database,target gene prediction and signal pathway analysis were performed by starBase,NONCODE,KEGG and other databases.The results showed that after infection of PK15 cells by JEV,the viral titer increased exponentially from 24 to 36 h,and the virus titer reached 10^-5.75 TCID₅₀/mL at 36 h after infection.There was no significant change in the expression of lncRNA A,B and C in PK15 cells after 12 h of infection (P>0.05),and the expression level of lncRNA D was extremely significantly decreased (P<0.01).The expression levels of lncRNA A,B and C increased extremely significantly after 24,36 and 48 h of infection (P<0.01),and the expression level of lncRNA D extremely significantly decreased (P<0.01).lncRNA A was mainly localized in the cytosol,lncRNA B was distributed in the nucleus and cytoplasm,lncRNA C was mainly located in the cytoplasm and might also be distributed in the nucleus,lncRNA D might be widely distributed in the cell.Through target gene prediction and signal pathway analysis,the target genes of lncRNA A,B and C were mainly OAS1,OAS2,OASL and COX1,etc.The target genes of lncRNA D were mainly DST,ND1,ND2,ND4 and so on.Signal pathway analysis revealed that lncRNA might participate in the proliferation process after viral infection through signaling pathways such as TNF,NF-κB and TLR.This study laid a foundation for further exploration of the effect of host cell lncRNA on viral proliferation.

Key words: lncRNA; PK15 cells; Japanese encephalitis virus (JEV); proliferation

中图分类号:

S828

杜程涛, 汪涵, 杨松柏, 李向臣, 周晓龙, 赵阿勇. lncRNA在乙脑病毒感染PK15细胞过程中的作用研究[J]. 《中国畜牧兽医》, 2019, 46(7): 2045-2052.

DU Chengtao, WANG Han, YANG Songbai, LI Xiangchen, ZHOU Xiaolong, ZHAO Ayong. Study on the Role of lncRNA in the Process of Japanese Encephalitis Virus Infecting PK15 Cells[J]. , 2019, 46(7): 2045-2052.

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