Preliminary Study On The Interaction Mechanism Between LncRNA NEAT1 And PRRSV

Porcine reproductive and respiratory syndrome(PRRS)is one of the most economically devastating diseases in the swine industry worldwide,which was first observed almost simultaneously in North America and Europe in the late 1980 s.Porcine reproductive and respiratory syndrome virus(PRRSV),the causative agent of PRRS,is an enveloped,single-stranded positive-sense RNA virus belonging to the order Nidovirales and family Arteriviridae.The PRRSV genome(?15kb)encodes at least 12 non-structural and seven structural proteins.Over the past decades,accumulating research evidence has shown that host lnc RNA plays a crucial role in tumor formation?immune regulation and virus replication.The NEAT1 is one of the most outstanding genes,and in addition to being involved in the regulation of immunology?inflammation and tumor formation,the NEAT1 also plays an extremely important role in the replication and pathogenesis of HIV-1?HSV-1? ZIKV and other viruses.However,the NEAT1 regulation mechanism of the host NEAT1 participating in PRRSV is not clear.Our study aims to scientifically reveal the interaction mechanism between lnc RNA NEAT1 gene and PRRSV,fill in new content for host-PRRSV interaction research from the lnc RNA level,providing ideas and clues for the discovery of other vital lnc RNA and their biological roles in the PRRSV infection process.To investigate the relationship between PRRSV infection and host NEAT1 transcription,MARC-145 cells were infected with PRRSV with MOI of 1,and RT-q PCR was set up to detect changes of NEAT1 at 12 time points.The results showed that NEAT1 transcription level was significantly increased after PRRSV-infected cells for 48 hours,and fluorescence in situ hybridization(FISH)also confirmed that PRRSV-infected cells can cause up-regulation of NEAT1 transcription.In addition,FISH also found that NEAT1 is fully localized in the nucleus.When cells were infected with viruses with MOIs of 0.1,0.5,1 and 2 and nucleic acids were extracted at 48 h,We found a significant difference in the transcriptional level of NEAT1 between the infected and uninfected groups,but there was no significant difference between the different MOIs,indicating that NEAT1 expression is not associated with PRRSV dose.To elucidate the role of NEAT1 in the PRRSV infection process,we obtained the full length of NEAT1(?23 kb)by RT-PCR,5'RACE and 3'RACE.The NEAT1 overexpression plasmid was constructed.Next,NEAT1 over-expression plasmids were transfected,and 12 hours later,cells were infected with PRRSV with MOI of 0.01.After 36 hours,RT-q PCR results showed that PRRSV replication levels were significantly reduced after overexpression of NEAT1.It is suggested that the rise of NEAT1 transcription level can inhibit PRRSV replication.To further explore how NEAT1 affects PRRSV replication,we inoculated PRRSV with MOI of 0.01 after overexpressing NEAT1,to observe changes in immune and inflammatory factors.IL-8 and IL-10 were significantly down-regulated after cell overexpression.The two target genes c-myc and cyclin D1 were also significantly reduced.It shows that NEAT1 does not prevent viral replication by regulating IL-8 and IL-10,and whether the NEAT1 will function through the WNT pathway will be explored in the following experiments.In order to clarify how PRRSV infection causes up-regulation of NEAT1,we inactivated the nucleic acids by means of ultraviolet light(254 nm),and irradiate PRRSV for 1 hour on ice to infect MARC-145 cells.It was found that UV-inactivated PRRSV did not induce up-regulation of NEAT1,The NEAT1 transcription level will not be raised due to the structural protein of PRRSV.To ensure nucleic acids and non-structural proteins,we used poly I:C(double-stranded RNA)to mimic the characteristics of PRRSV nucleic acids and transfect cells,and NEAT1 remained unchanged.It suggested that non-structural proteins play an important role in causing upregulation of NEAT1 transcription,which unstructured proteins have not been identified.To identify key pathways for PRRSV-induced upregulation of NEAT1,we designed si RNA targeting TLR-3 and MDA-5 signaling pathways and detected interference efficiencies of more than 50%.Based on RT-q PCR results,we found that NEAT1 expression was significantly reduced when the TLR-3 and MDA-5 pathways were knock-down and infected with PRRSV.It is suggested that PRRSV acts on NEAT1 transcription levels through these two pathways.In conclusion,we obtained the whole sequence of the MARC-145 NEAT1 is 23417 bp,the NEAT1 from MARC-145 cells is located in the nucleus,PRRSV acts on TLR3 and MDA-5 pathways through its nonstructural proteins to induce the rise of NEAT1.In addition,the rise of NEAT1 transcription level can inhibit the replication of PRRSV.