The Effect Of Long Non-coding RNA LNC＿000641 On PRV Replication And Its Molecular Mechanism

Pseudorabies(PR)is an acute infectious disease caused by pseudorabies virus(PRV).In the 1960 s,PR was firstly reported in China.To control the spread of PRV,the classic vaccine based on the strain Bartha-K61 were used to immune all of pigs.Since 2010,largescale outbreaks of Pseudorabies caused by the PRV variant strains have appeared in China.Vaccine is not effective in protecting pigs against infection of mutant strain.Therefore,it is of great significance to strengthen the research on the pathogenesis and immune regulation of PRV for the prevention and control of pseudorabies.Long non-coding RNA is a class of transcripts with more than 200 bp in length without encoding possibility in eukaryotes.Studies have shown that host lnc RNAs can regulate the replication of a variety of viruses.However,the effect of lnc RNA on PRV replication and its molecular mechanism are still unclear.In this study,the host lnc RNAs up-regulated after PRV infection in 3D4/21 cells were screened through RNA-sequence analysis and further verified by q RT-PCR.The effect of LNC＿000641 on PRV replication and its mechanism were studied by various methods.This research provides new insights for future PRV prevention and treatment strategies.The main contents of this research are as follows:1.RNA-sequence analysis of 3D4/21 cells infected with PRVTo identify the role of lnc RNA in pseudorabies virus infection and pathogenic mechanism,3D4/21 cells infected with PRV-ZJ01 strain at a MOI of 0.5 for 22 h were submitted to RNA-sequence analysis.Compared with control cells,225 lnc RNAs in PRVinfected 3D4/21 cells were significantly differentially expressed.Of them,126 1nc RNAs were up-regulated and 99 1nc RNAs were down-regulated.Compared with other studies and transcriptome databases,5 lnc RNAs were selected for further study.3D4/21 cells infected with PRV were detected by q RT-PCR,and the results showed that the 5 lnc RNAs were stably up-regulated in infected cells.In order to study the effect of virus strains,dose and infection time on the expression of lnc RNAs,q RT-PCR was used to detect cell samples infected with different strains,different doses and different times.The results showed that the expression of 5 lnc RNAs was up-regulated in infected cells regardless of virus strains,infection dose and infection time.In order to exclude the host singularity of these lnc RNAs,the same study was conducted in PK-15 cells and ST cells in this study.The results showed that the expression of 5 lnc RNAs in PK-15 cells and ST cells infected with PRV was consistent with that in 3D4 / 21 cells,indicating that these lnc RNAs were related to PRV infection in different cells.2.The effect of LNC＿000641 on PRV replicationTo identify the effect of lnc RNAs on PRV replication and its molecular mechanism,this study investigated the effect of LNC＿000641 on PRV replication.First,the recombinant plasmid and si RNA interference fragments were used to regulate the expression of LNC＿000641,and then Western blot,q RT-PCR,IFA,and TCID50 were used to detect the expression of LNC＿000641 and PRV replication.The results showed that LNC＿000641 significantly promoted the replication of PRV in 3D4/21 cells in a dosedependent manner.3.The molecular mechanism of LNC＿000641 regulating the proliferation of pseudorabies virusTo elucidate the mechanisms of LNC＿000641 mediated PRV replication,the IFNalpha expression level in cells regulated the expression of LNC＿000641 was determined.The results showed that,IFN-alpha m RNA expression in cells silenced LNC＿000641increased significantly.At the same time,overexpression of LNC＿000641 inhibited the m RNA of IFN-alpha.To elucidate the mechanisms of LNC＿000641 mediated IFN-alpha expression,the total protein and phosphorylation levels of JAK and STAT1 were detected by Western blot.The results showed that the phosphorylated JAK and STAT1 decreased significantly after over-expression of LNC＿000641.At the same time,the phosphorylated JAK and STAT1 were significantly increased after knocking down LNC＿000641.The results indicated that LNC＿000641 regulates IFN-alpha expression and then affects the JAK/STAT1 pathway and thus regulates PRV replication.This study revealed for the first time that LNC＿000641 can regulate PRV replication,and found that its molecular mechanism may be related to the JAK/STAT1 pathway.This research provides new insights for future PRV prevention and treatment strategies.