Analysis Of Transcriptome Of PRV LncRNAs Expressed In Epithelial Cells And Expression Of These RNAs In Neuron Cells

Long noncoding RNAs(lncRNAs)are a kind of single-stranded RNA molecules of which length are more than 200 bp.Most lncRNAs do not encode anmino acid sequces except for a few lncRNAs that can be translated into peptides.In eukaryotic cells,lncRNAs are multifunctional regulators,including as decoys to sequenster proteins or nucleotides to interact with their substances,as scaffolds to form complexes with other components and as guides to make some proteins achieve their subcellular localization.LncRNAs involve individual's growth,development,immunity,epigenetics and self-related diseases by participating in cell's replicaton,growth,differentiation,apoptosis and so on.Pseudorabies virus(PRV)is a pathogen belonging to alpha herpsvirus family.The infection of this pathogen results in devastating diseases on pigs and enomous economic losses all over the world.When PRV infects its natural hosts,latent infection is caused in centry nervous system before the virus replicates in epithelial cells followed by invading neurons around epithelial cells.In latent infection,PRV transcribes several high abundant lantency-associated transcripts(LATs)between ep0 and ie180.LATs are lncRNAs and some LATs can be detected in productive infection in epithelial cells.Thus,there might be other lncRNAs transcribed by PRV in the infection of epithelial cells and these lncRNA may play an important role in PRV's infection.The structures of most lncRNAs resemble eukaryotic mRNA with both 5? cap and 3? ploy(A),but a few lncRNAs only contain 5? cap or 3? ploy(A),or neither 5? cap nor 3? ploy(A).In order to discover all lncRNAs in PRV-infected PK-15 epithelial cells,random hemaxers were used in reverse transcription followed by constructing strand-specific library.Data from high-throughput sequencing was analysed by bioinformatics to identify lncRNAs from PRV or epithelial cells.Then lncRNAs transcribed from PRV or epithelial cells were validated by RT-PCR,northern blot and/or 3? RACE.The expression of validated lncRNA was aslo demonstrated by real-time PCR in both PK-15 cells and primary chicken embryo dorsal root ganglion(DRG)cells infected by PRV.Finally,we sued siRNAs to interfere viral lncRNAs' expression in PK-15 cell and the viral titers from siRNA interfering significantly increased.Therefore,our results provided a foundation and a new sight for PRV latent infection involved by lncRNA.The main work of this study consists of following parts: 1.Transcriptomics sequencing of lncRNAs and analysis of sequencing dataWe isolated total RNA from PK-15 cells infected by PRV with 1 MOI after 24 hpi.Qualitified RNA was used for constructing strand-specific library followed by sequencing with Illumina Hiseq2500 system.We identified 28262 lncRNAs in total from sequencing data,including 3 novel viral lnc RNAs,28224 novel host lncRNAs and 35 known host lncRNAs.2.Demonstrating lncRNAs by RT-PCR,northern blot and 3? RACEOf all lncRNAs,one known viral lncRNA,two novel viral lncRNAs and five novel host lncRNAs were demonstrated by RT-PCR.Northern blot result showed only one lncRNA can be specifically detected whereas others were unspecific signals.We also obtained the 3? end sequence of one known viral lncRNA by 3? RACE.3.Validation of viral lncRNAs from PRV-infected neuron cells by real-time PCRWe successfully isolated primary dorsal root ganglion(DRG)cells from 10-days SPF chicken embryos,and cultivated these cells in plates or microfluidic devices.Then matural neuron cells cultured in plates were infected by PRV.All three viral lncRNAs were validated by real-time PCR and RT-PCR and the results displayed that the expression abundance of three lncRNAs was dependent on infected times and viral amounts when PRV infected neuron cells.We aslo built a neuron-infected model of PRV in microfluidic devices by co-culturing neuron cells and PK-15 cells.And qPCR results showed the expression abundance of three lnc RNAs was different betweent infected neuron cells cultivated in plates and microfluidic devices.4.Effects of PRV-transcribed lncRNAs on viral replicationThree novel viral lncRNAs were interfered by siRNAs when PRV infected PK-15 cells.From the results of viral titers after 12 hpi,siRNA interfering of two lncRNAs significantly promoted the replication of PRV.Collectively,our results show that PRV infection doesn't noly induce PRV transcribes lncRNAs from its genome,but also stimulates host cells generate a number of lncRNAs and restrains the expression of a mass of host lncRNAs.These viral lncRNAs also were producted in neuron cells cultivated in plates or microfluidic devices.And siRNA interfering against viral lncRNAs can increase viral titers.