lncRNA在乙脑病毒感染PK15细胞过程中的作用研究
|
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TCID_(50)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) TCID_(50)/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。
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 TCID_(50) 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) TCID_(50)/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.
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 TCID_(50) 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) TCID_(50)/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.
引文
[1] CHAI C,WANG Q,CAO S,et al.Serological and molecular epidemiology of Japanese encephalitis virus infections in swine herds in China[J].Journal of Veterinary Science,2018,19(1):151-155.
[2] ZHANG Y,JING J,LI X,et al.Integration analysis of miRNA and mRNA expression profiles in swine testis cells infected with Japanese encephalitis virus[J].Infection,Genetics and Evolution,2015,32:342-347.
[3] FAN J M,LUO J,CHEN L,et al.Genetic analysis of strains of Japanese encephalitis virus isolated from swine in central China[J].Virus Genes,2010,40(3):357-361.
[4] YUN S I,LEE Y M.Japanese encephalitis:The virus and vaccines[J].Human Vaccines & Immunotherapeutics,2014,10(2):263-279.
[5] ZHU Y Z,XU Q Q,WU D G,et al.Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent,dynamin and caveola-mediated endocytosis pathway[J].Journal of Virology,2012,86(24):13407-13422.
[6] KALIA M,KHASA R,SHARMA M,et al.Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism[J].Journal of Virology,2013,87(1):148-162.
[7] YANG S,HE M H,LIU X D,et al.Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin- and cholesterol-dependent endocytosis[J].Virology Journal,2013,10:258.
[8] CHUANG C K,YANG T H,CHEN T H,et al.Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells[J].Journal of General Virology,2015,96(Pt 4):793-803.
[9] NAWA M.Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug,chlorpromazine[J].Journal of General Virology,2003,84(7):1737-1741.
[10] YANG S B,PEI Y,LI X,et al.miR-124 attenuates Japanese encephalitis virus replication by targeting DNM2[J].Virology Journal,2016,13:105.
[11] LIU C C,ZHANG Y N,LI Z Y,et al.Rab5 and Rab11 are required for clathrin-dependent endocytosis of Japanese encephalitis virus in BHK-21 cells[J].Journal of Virology,2017,91(19):e01113-17.
[12] LAURENT G S,WAHLESTEDT C,KAPRANOV P.The landscape of long noncoding RNA classification[J].Trends in Genetics Tig,2015,31(5):239-251.
[13] PONTING C P,OLIVER P L,REIK W.Evolution and functions of long noncoding RNAs[J].Cell,2009,136(4):629-641.
[14] LIU W W,CHAN D.Roles of lncrnas in viral infections[J].Frontiers in Cellular and Infection Microbiology,2017,7:205.
[15] CARPENTER S,AIELLO D,ATIANAND M K,et al.A long noncoding RNA mediates both activation and repression of immune response genes[J].Science,2013,341(6147):789-792.
[16] CHEN C J,CHEN J H,CHEN S Y,et al.Upregulation of RANTES gene expression in neuroglia by Japanese encephalitis virus infection[J].Journal of Virology,2004,78(22):12107-12119.
[17] SMITH J A,DAS A,RAY S K,et al.Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases[J].Brain Research Bulletin,2012,87(1):10-20.
[18] CHEN Z,YE J,ASHRAF U,et al.Modulates Japanese encephalitis virus replication by targeting eukaryotic translation elongation factor 1A1[J].Journal of Virology,2016,90(7):3722-3734.
[19] ASHRAF U,ZHU B,YE J,et al.microRNA-19b-3p modulates Japanese encephalitis virus-mediated inflammation via targeting RNF11[J].Journal of Virology,2016,90(9):4780-4795.
[20] CARDOSO A L,GUEDES J R,LIMA M C P D.Role of microRNAs in the regulation of innate immune cells under neuroinflammatory conditions[J].Current Opinion in Pharmacology,2016,26:1-9.
[21] RASTOGI M,SRIVASTAVA N,SINGH S K.Exploitation of microRNAs by Japanese encephalitis virus in human microglial cells[J].Journal of Medical Virology,2017,90(4):648-654.
[22] BHATTACHARYYA S,VRATI S.The malat1 long non-coding RNA is upregulated by signalling through the PERK axis of unfolded protein response during flavivirus infection[J].Scientific Reports,2015,5(11):17794.
[23] LI Y,ZHANG H,ZHU B,et al.Microarray analysis identifies the potential role of long non-coding RNA in regulating neuroinflammation during Japanese encephalitis virus infection[J].Frontiers in Immunology,2017,8:1237.
[24] YANG Y,YE J,YANG X,et al.Japanese encephalitis virus infection induces changes of mRNA profile of mouse spleen and brain[J].Virology Journal,2011,8:80.
[25] PAREEK S,ROY S,KUMARI B,et al.miR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses[J].Journal of Neuroinflammation,2014,11(1):97.
[26] THOUNAOJAM M C,KAUSHIK D K,KUNDU K,et al.MicroRNA-29b modulates Japanese encephalitis virus-induced microglia activation by targeting tumor necrosis factor alpha-induced protein 3[J].Journal of Neurochemistry,2014,129(1):143-154.
[27] TAO X W,ZENG L K,WANG H Z,et al.LncRNA MEG3 ameliorates respiratory syncytial virus infection by suppressing TLR4 signaling[J].Molecular Medicine Reports,2018,17(3):4138-4144.
[28] ZHANG J,SUN P,GAN L,et al.Genome-wide analysis of long noncoding RNA profiling in PRRSV-infected PAM cells by RNA sequencing[J].Scientific Reports,2017,7(1):4952.
[29] WANG X J,JIANG S C,WEI H X,et al.The differential expression and possible function of long noncoding RNAs in liver cells infected by dengue virus[J].The American Journal of Tropical Medicine and Hygiene,2017,97(6):1904-1912.
[30] FU N,ZHAO S X,KONG L B,et al.LncRNA-ATB/microRNA-200a/β-catenin regulatory axis involved in the progression of HCV-related hepatic fibrosis[J].Gene,2017,618:1-7.
[31] KOTZIN J J,MOWEL W K,HENAOMEJIA J.Viruses hijack a host lncRNA to replicate[J].Science,2017,358(6366):993-994.
[32] WANG P,XU J,WANG Y,et al.An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism[J].Science,2017,358(6366):1051-1055.
[33] CHIYOMARU T,YAMAMURA S,FUKUHARA S,et al.Genistein inhibits prostate cancer cell growth by targeting miR-34a and oncogenic HOTAIR[J].PLoS One,2013,8(8):e70372.
[34] 郑伟.LncRNA-miRNA-mRNA相互作用初步研究[D].北京:中国人民解放军军事医学科学院,2017.ZHENG W.Preliminary study on the interaction of LncRNA-miRNA-mRNA[D].Beijing:Chinese Academy of Military Medical Sciences,2017.(in Chinese)
[35] JIA L F,WEI S B,GAN Y H,et al.Expression,regulation and roles of miR-26a and MEG3 in tongue squamous cell carcinoma[J].International Journal of Cancer,2014,135(10):2282-2293.
[36] LIU K,LIAO X,ZHOU B,et al.Porcine alpha interferon inhibit Japanese encephalitis virus replication by different ISGs in vitro[J].Research in Veterinary Science,2013,95(3):950-956.
[37] SWARUP V,GHOSH J,GHOSH S,et al.Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis[J].Antimicrobial Agents and Chemotherapy,2007,51(9):3367-3370.
[2] ZHANG Y,JING J,LI X,et al.Integration analysis of miRNA and mRNA expression profiles in swine testis cells infected with Japanese encephalitis virus[J].Infection,Genetics and Evolution,2015,32:342-347.
[3] FAN J M,LUO J,CHEN L,et al.Genetic analysis of strains of Japanese encephalitis virus isolated from swine in central China[J].Virus Genes,2010,40(3):357-361.
[4] YUN S I,LEE Y M.Japanese encephalitis:The virus and vaccines[J].Human Vaccines & Immunotherapeutics,2014,10(2):263-279.
[5] ZHU Y Z,XU Q Q,WU D G,et al.Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent,dynamin and caveola-mediated endocytosis pathway[J].Journal of Virology,2012,86(24):13407-13422.
[6] KALIA M,KHASA R,SHARMA M,et al.Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism[J].Journal of Virology,2013,87(1):148-162.
[7] YANG S,HE M H,LIU X D,et al.Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin- and cholesterol-dependent endocytosis[J].Virology Journal,2013,10:258.
[8] CHUANG C K,YANG T H,CHEN T H,et al.Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells[J].Journal of General Virology,2015,96(Pt 4):793-803.
[9] NAWA M.Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug,chlorpromazine[J].Journal of General Virology,2003,84(7):1737-1741.
[10] YANG S B,PEI Y,LI X,et al.miR-124 attenuates Japanese encephalitis virus replication by targeting DNM2[J].Virology Journal,2016,13:105.
[11] LIU C C,ZHANG Y N,LI Z Y,et al.Rab5 and Rab11 are required for clathrin-dependent endocytosis of Japanese encephalitis virus in BHK-21 cells[J].Journal of Virology,2017,91(19):e01113-17.
[12] LAURENT G S,WAHLESTEDT C,KAPRANOV P.The landscape of long noncoding RNA classification[J].Trends in Genetics Tig,2015,31(5):239-251.
[13] PONTING C P,OLIVER P L,REIK W.Evolution and functions of long noncoding RNAs[J].Cell,2009,136(4):629-641.
[14] LIU W W,CHAN D.Roles of lncrnas in viral infections[J].Frontiers in Cellular and Infection Microbiology,2017,7:205.
[15] CARPENTER S,AIELLO D,ATIANAND M K,et al.A long noncoding RNA mediates both activation and repression of immune response genes[J].Science,2013,341(6147):789-792.
[16] CHEN C J,CHEN J H,CHEN S Y,et al.Upregulation of RANTES gene expression in neuroglia by Japanese encephalitis virus infection[J].Journal of Virology,2004,78(22):12107-12119.
[17] SMITH J A,DAS A,RAY S K,et al.Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases[J].Brain Research Bulletin,2012,87(1):10-20.
[18] CHEN Z,YE J,ASHRAF U,et al.Modulates Japanese encephalitis virus replication by targeting eukaryotic translation elongation factor 1A1[J].Journal of Virology,2016,90(7):3722-3734.
[19] ASHRAF U,ZHU B,YE J,et al.microRNA-19b-3p modulates Japanese encephalitis virus-mediated inflammation via targeting RNF11[J].Journal of Virology,2016,90(9):4780-4795.
[20] CARDOSO A L,GUEDES J R,LIMA M C P D.Role of microRNAs in the regulation of innate immune cells under neuroinflammatory conditions[J].Current Opinion in Pharmacology,2016,26:1-9.
[21] RASTOGI M,SRIVASTAVA N,SINGH S K.Exploitation of microRNAs by Japanese encephalitis virus in human microglial cells[J].Journal of Medical Virology,2017,90(4):648-654.
[22] BHATTACHARYYA S,VRATI S.The malat1 long non-coding RNA is upregulated by signalling through the PERK axis of unfolded protein response during flavivirus infection[J].Scientific Reports,2015,5(11):17794.
[23] LI Y,ZHANG H,ZHU B,et al.Microarray analysis identifies the potential role of long non-coding RNA in regulating neuroinflammation during Japanese encephalitis virus infection[J].Frontiers in Immunology,2017,8:1237.
[24] YANG Y,YE J,YANG X,et al.Japanese encephalitis virus infection induces changes of mRNA profile of mouse spleen and brain[J].Virology Journal,2011,8:80.
[25] PAREEK S,ROY S,KUMARI B,et al.miR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses[J].Journal of Neuroinflammation,2014,11(1):97.
[26] THOUNAOJAM M C,KAUSHIK D K,KUNDU K,et al.MicroRNA-29b modulates Japanese encephalitis virus-induced microglia activation by targeting tumor necrosis factor alpha-induced protein 3[J].Journal of Neurochemistry,2014,129(1):143-154.
[27] TAO X W,ZENG L K,WANG H Z,et al.LncRNA MEG3 ameliorates respiratory syncytial virus infection by suppressing TLR4 signaling[J].Molecular Medicine Reports,2018,17(3):4138-4144.
[28] ZHANG J,SUN P,GAN L,et al.Genome-wide analysis of long noncoding RNA profiling in PRRSV-infected PAM cells by RNA sequencing[J].Scientific Reports,2017,7(1):4952.
[29] WANG X J,JIANG S C,WEI H X,et al.The differential expression and possible function of long noncoding RNAs in liver cells infected by dengue virus[J].The American Journal of Tropical Medicine and Hygiene,2017,97(6):1904-1912.
[30] FU N,ZHAO S X,KONG L B,et al.LncRNA-ATB/microRNA-200a/β-catenin regulatory axis involved in the progression of HCV-related hepatic fibrosis[J].Gene,2017,618:1-7.
[31] KOTZIN J J,MOWEL W K,HENAOMEJIA J.Viruses hijack a host lncRNA to replicate[J].Science,2017,358(6366):993-994.
[32] WANG P,XU J,WANG Y,et al.An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism[J].Science,2017,358(6366):1051-1055.
[33] CHIYOMARU T,YAMAMURA S,FUKUHARA S,et al.Genistein inhibits prostate cancer cell growth by targeting miR-34a and oncogenic HOTAIR[J].PLoS One,2013,8(8):e70372.
[34] 郑伟.LncRNA-miRNA-mRNA相互作用初步研究[D].北京:中国人民解放军军事医学科学院,2017.ZHENG W.Preliminary study on the interaction of LncRNA-miRNA-mRNA[D].Beijing:Chinese Academy of Military Medical Sciences,2017.(in Chinese)
[35] JIA L F,WEI S B,GAN Y H,et al.Expression,regulation and roles of miR-26a and MEG3 in tongue squamous cell carcinoma[J].International Journal of Cancer,2014,135(10):2282-2293.
[36] LIU K,LIAO X,ZHOU B,et al.Porcine alpha interferon inhibit Japanese encephalitis virus replication by different ISGs in vitro[J].Research in Veterinary Science,2013,95(3):950-956.
[37] SWARUP V,GHOSH J,GHOSH S,et al.Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis[J].Antimicrobial Agents and Chemotherapy,2007,51(9):3367-3370.