长链非编码RNA在PEDV感染Vero-E6细胞中对自噬的调控作用  

Regulation of Long Noncoding RNAs in Autophagy Induced by PEDV Infected Vero-E6 Cells

作　　者：王梓行 邓兴梅 邱润辉 朱德馨 李佳 陶婷婷 朱嘉乐 孙志华[1,2,3] 张辉 WANG Zihang;DENG Xingmei;QIU Runhui;ZHU Dexin;LI Jia;TAO Tingting;ZHU Jiale;SUN Zhihua;ZHANG Hui(College of Animal Science and Technology,Shihezi 832000,China;Key Laboratory of Control and Prevention of Animal Disease,Xinjiang Production&Construction Corps,Shihezi 832000,China;State International Joint Research Center for Animal HealthBreeding,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,石河子832000 [2]新疆生产建设兵团动物疾病防控重点实验室,石河子832000 [3]动物健康养殖国家国际联合研究中心,石河子832000

出　　处：《中国畜牧兽医》2022年第5期1942-1950,共9页China Animal Husbandry & Veterinary Medicine

基　　金：重点领域科技攻关计划(2021AB012);国际科技合作推进计划(GJHZ201709);动物疾病防控兵团重点实验室开放课题(2020BTDJ06);石河子大学高层次人才科研启动项目(RCZK202038)。

摘　　要：【目的】试验旨在研究长链非编码RNA(long noncoding RNA,lncRNA)在猪流行性腹泻病毒(Porcine epidemic diarrhea viru,PEDV)感染非洲绿猴肾细胞(Vero-E6)过程中对自噬的调控作用,以期为抗PEDV新型药物靶点的开发提供新思路。【方法】根据GenBank上公布的人lncRNA-HOTAIR基因序列(登录号:NR_047517.1),利用LongMan在线工具筛选lncRNA-HOTAIR的猴源同源序列(lncRNA-M),并利用RNAfold、LncLocator在线预测工具预测其二级结构和核质分布。建立PEDV感染的Vero-E6细胞模型,在0、6、12、24、36和48 h收集细胞,利用实时荧光定量PCR和Western blotting检测微管相关蛋白1-轻链3(LC3)蛋白的表达,以明确病毒感染细胞后不同时间点自噬的发生情况。通过实时荧光定量PCR检测lncRNA-M在PEDV感染Vero-E6细胞模型中0、6、12、24、36和48 h的表达水平。设计并合成3条lncRNA-M的干扰片段:siRNA-1、siRNA-2和siRNA-3,分别转染Vero-E6细胞,待细胞汇合度达到90%时接毒,感染48 h后利用实时荧光定量PCR检测lncRNA-M的表达情况,筛选出干扰效率最高的干扰片段。将干扰效率最高的干扰片段转染至Vero-E6细胞并接种PEDV后,通过Western blotting检测感染后24和48 h LC3蛋白的表达水平,以验证自噬的发生情况。【结果】成功筛选出lncRNA-HOTAIR猴源同源序列为lncRNA 0.1,并根据RNAfold预测结果生成lncRNA 0.1的RNA最小自由能二级结构;LncLocator预测结果表明,lncRNA 0.1在细胞核和细胞质中的分布分别为41.88%和37.78%。PEDV感染Vero-E6细胞48 h后,细胞皱缩、聚集成团,细胞膜融合形成合胞体;1.0%琼脂糖凝胶电泳结果显示,在1326 bp处出现目的条带,说明PEDV感染Vero-E6细胞模型成功建立。在PEDV感染的Vero-E6细胞模型中,Western blotting结果显示,6、12、24、36和48 h LC3Ⅱ的表达量呈上升趋势,LC3Ⅱ/LC3Ⅰ的比值在48 h最高且极显著高于0 h(P<0.01);实时荧光定量PCR结果显示,LC3的mRNA表达量在48 h最高,且极【Objective】The purpose of the experiment was to study the regulation of long noncoding RNA(lncRNA)on autophagy in the process of Porcine epidemic diarrhea virus(PEDV)infecting African green monkey kidney cells(Vero-E6),so as to provide a new idea for the development of new drug targets against PEDV.【Method】According to the human published lncRNA-HOTAIR gene sequence(accession No.:NR_047517.1)in GenBank,the monkey homologous sequence of lncRNA-HOTAIR(lncRNA-M)was screened by LongMan online tool,and the secondary structure and nucleocytoplasmic distribution were predicted by RNAfold and LncLocator online prediction tools.The Vero-E6 cell model infected by PEDV was established.The cells were collected at 0,6,12,24,36 and 48 h,and Real-time quantitative PCR was used to detect microtubule associated protein 1 light chain 3(LC3),in order to determine the occurrence of autophagy at different time points after virus infected cells.The expression levels of lncRNA-M in Vero-E6 cell model infected by PEDV at 0,6,12,24,36 and 48 h were detected by Real-time quantitative PCR.Three lncRNA-M interference fragments siRNA-1,siRNA-2 and siRNA-3 were designed,synthesized and transfected into Vero-E6 cells respectively.When the confluence of Vero-E6 cells reached 90%,the cells were infected with PEDV,and after infection for 48 h,the expression of lncRNA-M was detected by Real-time quantitative PCR,and the interference fragment with the highest interference efficiency was screened.After transfecting the interference fragment with the highest interference efficiency into Vero-E6 cells,and the cells were infected with PEDV,the expression level of LC3 protein at 24 and 48 h after infection was detected by Western blotting to verify the occurrence of autophagy.【Result】The homologous sequence of monkey lncRNA-HOTAIR was successfully screened as lncRNA 0.1,and the RNA minimum free energy secondary structure of lncRNA 0.1 was generated according to the prediction results of RNAfold.The prediction results of Lnlocator showed that

关 键 词：猪流行性腹泻病毒(PEDV) 自噬 长链非编码RNA LC3蛋白 

分 类 号：S852.659.2[农业科学—基础兽医学]