长链非编码RNA在PEDV感染Vero-E6细胞中对自噬的调控作用

doi:10.16431/j.cnki.1671-7236.2022.05.035

摘要/Abstract

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1 326 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，且极显著高于0 h(P<0.01)，自噬被激活;lncRNA 0.1的表达均呈上升趋势，在48 h表达量最高，48 h lncRNA 0.1的表达量极显著高于0 h (P<0.01);siRNA-2的干扰效果最好，干扰效率为80%。干扰lncRNA 0.1后，Western blotting结果显示，24和48 h干扰组的LC3蛋白表达量均低于对照组，自噬受到抑制。【结论】 PEDV感染Vero-E6细胞能够诱导自噬发生，lncRNA 0.1在感染过程中起到了促进自噬的作用。

关键词: 猪流行性腹泻病毒(PEDV); 自噬; 长链非编码RNA; LC3蛋白

Abstract: 【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 the distribution of lncRNA 0.1 in nucleus and cytoplasm were 41.88% and 37.78%,respectively.48 h after PEDV infected Vero-E6 cells,the cells shrank and clustered,and the cell membrane fused to form syncytia body.1.0% agarose gel electrophoresis showed that the target band appeared at 1 326 bp,indicating that the PEDV infection Vero-E6 cell model was successfully established.In Vero-E6 cell model infected by PEDV,Western blotting results showed that the expression of LC3Ⅱ was increased in the experimental group at 6,12,24,36 and 48 h,the ratio of LC3Ⅱ /LC3Ⅰ was the highest at 48 h and was extremely significantly higher than that of control group (P<0.01).Real-time quantitative PCR results showed that the mRNA expression of LC3 was the highest at 48 h and was extremely significantly higher than that of control group (P<0.01),autophagy was activated.The expression of lncRNA 0.1 showed an upward trend,and the expression was the highest at 48 h,and the expression of lncRNA 0.1 at 48 h test group was extremely significantly higher than that in control group (P<0.01).Real-time quantitative PCR showed that siRNA-2 had the best interference effect,and the interference efficiency was 80%.After interfering with lncRNA 0.1,Western blotting results showed that the expression of LC3 protein was decreased and autophagy was inhibited in 24 and 48 h interference groups.【Conclusion】 PEDV infection could induce Vero-E6 autophagy,and lncRNA 0.1 played a role in promoting autophagy during infection.

Key words: Porcine epidemic diarrhea virus(PEDV); autophagy; long noncoding RNA; LC3 protein

中图分类号:

S852.65⁺9.2

王梓行, 邓兴梅, 邱润辉, 朱德馨, 李佳, 陶婷婷, 朱嘉乐, 孙志华, 张辉. 长链非编码RNA在PEDV感染Vero-E6细胞中对自噬的调控作用[J]. 中国畜牧兽医, 2022, 49(5): 1942-1950.

WANG Zihang, DENG Xingmei, QIU Runhui, ZHU Dexin, LI Jia, TAO Tingting, ZHU Jiale, SUN Zhihua, ZHANG Hui. Regulation of Long Noncoding RNAs in Autophagy Induced by PEDV Infected Vero-E6 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1942-1950.

参考文献

[1] 吴白,苏玮玮,鞠德财,等.猪流行性腹泻病毒SD201604株的分离鉴定及致病性研究[J].中国畜牧兽医,2018,45(11):3229-3236. WU B,SU W W,JU D C,et al.Isolation,identification and pathogenicity evaluation of Porcine epidemic diarrhea virus SD201604 strain[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3229-3236.(in Chinese)
[2] 吴雨,温志芬,韩明政,等.猪流行性腹泻病毒致病机理研究进展[J].动物医学进展,2020,41(11):108-112. WU Y,WEN Z F,HAN M Z,et al.Research progress on pathogenic mechanism of Porcine epidemic diarrhea virus[J].Progress in Veterinary Medicine,2020,41(11):108-112.(in Chinese)
[3] JUNG K,SAIF L J.Porcine epidemic diarrhea virus infection:Etiology,epidemiology,pathogenesis and immunoprophylaxis[J].The Veterinary Journal,2015,204(2):134-143.
[4] 郭思杰,刘俊琦,李玉娟,等.猪流行性腹泻及新型疫苗研究进展[J].中国畜牧兽医,2020,47(9):2935-2944. GUO S J,LIU J Q,LI Y J,et al.Research advances on porcine epidemic diarrhea and new vaccine[J].China Animal Husbandry & Veterinary Medicine,2020,47(9):2935-2944.(in Chinese)
[5] CHANDRA V,BHAGYARAJ E,PARKESH R,et al.Transcription factors and cognate signalling cascades in the regulation of autophagy[J].Biological Reviews of the Cambridge Philosophical Society,2016,91(2):429-451.
[6] KADOWAKI M,KARIM M.Cytosolic LC3 ratio as a quantitative index of macroautophagy[J].Methods in Enzymology,2009,452:199-213.
[7] MILLER K,MCGRATH M E,HU Z,et al.Coronavirus interactions with the cellular autophagy machinery[J].Autophagy,2020,16(12):2131-2139.
[8] 李小璟,龚双燕,李幽幽,等.猪繁殖与呼吸综合征病毒诱导Marc-145细胞自噬的超微结构[J].农业生物技术学报,2020,28(1):101-107. LI X J,GONG S Y,LI Y Y,et al.Ultrastructure of Marc-145 cells in autophagy induced by Porcine reproductive and respiratory syndrome virus[J].Journal of Agricultural Biotechnology,2020,28(1):101-107.(in Chinese)
[9] MENG S,JIANG K,ZHANG X,et al.Avian reovirus triggers autophagy in primary chicken fibroblast cells and Vero cells to promote virus production[J].Archives of Virology,2012,157(4):661-668.
[10] GUO X,ZHANG M,ZHANG X,et al.Porcine epidemic diarrhea virus induces autophagy to benefit its replication[J].Viruses,2017,9(3):53.
[11] LIANG L,HUAN L,WANG J,et al.lncRNA RP11-295G20.2 regulates hepatocellular carcinoma cell growth and autophagy by targeting PTEN to lysosomal degradation[J].Cell Discovery,2021,7(1):118.
[12] SUN D,WANG X,WEI S,et al.Epidemiology and vaccine of Porcine epidemic diarrhea virus in China:A mini-review[J].The Journal of Veterinary Medical Science,2016,78(3):355-363.
[13] 蒋新华,周荷田,罗锋,等.2017年江西部分地区PEDV分子流行病学调查[J].中国畜牧兽医,2019,46(5):1474-1482. JIANG X H,ZHOU H T,LUO F,et al.Molecular epidemiological investigation of Porcine epidemic diarrhea virus[J].China Animal Husbandry & Veterinary Medicine,2019,46(5):1474-1482.(in Chinese)
[14] SHI W,HAO H,LI M,et al.Expression and purification of a PEDV-neutralizing antibody and its functional verification[J].Viruses,2021,13(3):1472.
[15] LIN H,LI B,LIU M,et al.Nonstructural protein 6 of Porcine epidemic diarrhea virus induces autophagy to promote viral replication via the PI3K/Akt/mTOR axis[J].Veterinary Microbiology,2020,244:108684.
[16] JACKSON W T.Viruses and the autophagy pathway[J].Virology,2015,479-480:450-456.
[17] LIU W,WANG Z,LIU L,et al.lncRNA Malat1 inhibition of TDP43 cleavage suppresses IRF3-initiated antiviral innate immunity[J].Proceedings of the National Academy of Sciences of the United States of America,2020,117(38):23695-23706.
[18] WANG P,XU J,WANG Y,et al.An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism[J].Science (New York,N.Y.),2017,358(6366):1051-1055.
[19] 马齐蔓.BVDV感染MDBK细胞的lncRNA文库构建及lncRNA 1620调控细胞凋亡的研究[D].石河子:石河子大学,2018. MA Q M.Study on construction of lncRNA library of BVDV infected MDBK cells and the regulation of apoptosis by lncRNA 1620[D].Shihezi:Shihezi University,2018.(in Chinese)
[20] 王磊.猪流行性腹泻病毒影响感染细胞内调控NF-κB信号通路的lncRNA表达研究[D].杭州:浙江农林大学,2020. WANG L.Expression of lncRNA regulating NF-κB signaling pathway in PEDV infected cells[D].Hangzhou:Zhejiang A&F University,2020.(in Chinese)
[21] ZHANG J,CHEN K,TANG Y,et al.lncRNA-HOTAIR activates autophagy and promotes the imatinib resistance of gastrointestinal stromal tumor cells through a mechanism involving the miR-130a/ATG2B pathway[J].Cell Death & Disease,2021,12(4):367.
[22] JIA L,SONG Y,MU L,et al.Long noncoding RNA TPT1-AS1 downregulates the microRNA-770-5p expression to inhibit glioma cell autophagy and promote proliferation through STMN1 upregulation[J].Journal of Cellular Physiology,2020,235(4):3679-3689.
[23] YU L,DONG J,LIU Y,et al.Genome-wide analysis of long noncoding RNA profiles in Vero cells infected with Porcine epidemic diarrhea virus[J].Archives of Virology,2020,165(9):1969-1977.