The Role And Mechanism Of Long Non-coding RNA In The Apoptosis Of Host Cells Induced By BVDV Infection

Bovine viral diarrhea-mucosal disease is a febrile fever infectious disease caused by the bovine viral diarrhea virus(BVDV),which has a worldwide distribution and often causes persistent infections and immunosuppression in cattle.Inflammation,erosion,and necrosis,and concurrent respiratory infections are the main clinical features.The disease severely affects the performance of animals and causes significant economic losses to the cattle industry.Therefore,in-depth study of the pathogenic mechanism of BVDV is of great significance for the prevention and treatment of this disease.Long non-coding RNA(long non-coding RNA,lnc RNA)is widely involved in regulating the occurrence and development of various diseases.For example,lnc RNA participates in the interaction between the virus and the host in viral infectious diseases.The pathogenic process can also regulate host immunity to inhibit viral infection.Previous studies have shown that BVDV can induce apoptosis during the pathogenesis of BVDV infection.In order to further understand the molecular mechanism of BVDV-induced apoptosis,this study uses RNA-seq technology to analyze BVDV-infected host(MDBK)cells The subsequent lnc RNA differential expression profile was screened by KEGG signal pathway enrichment analysis and lnc RNA-m RNA coexpression network analysis to screen out lnc RNAs that regulate cell apoptosis,and studied its potential regulatory mechanism.First,in this study,RNA-seq technology was used for sequencing analysis to obtain the lnc RNA differential expression profile after BVDV infection of MDBK.The specific content is as follows: Total RNA of MDBK cells(experimental group)and non-infected MDBK cell(control group)were extracted 48 hours after infection with BVDV AV69 virus strain,and qualified RNA samples(3 parallel in each group)Samples and RNA samples with completeness> 9.8,absorbance 260/280> 2,total amount> 2 ?g)for library building and transcriptome sequencing analysis;qualified sequencing data was compared with the reference genome of cattle using software Top Hat 2.1.1 Then,Cufflinks were used to assemble transcripts,and CNCI,CPC,and PCA were used to predict lnc RNAs.Then,RSEM software and FPKM were used to calculate the expression of lnc RNAs and m RNAs in the experimental group relative to the control group.The results showed that a total of 1591 m RNAs with a fold difference> 1 and a p-value <0.05 were obtained,including 895 m RNAs with up-regulated expression and 696 m RNAs with down-regulated expression;a total of 156 differentially expressed lnc RNAs were obtained,of which up-regulated lnc RNAs 72 The number of differentially expressed lnc RNAs(BIRC2,IGF1 R,PTCD2,DTX2,MAPK8,and GTF2I)and the differentially expressed lnc RNAs(loc101906383,TCONS00026615,loc112444864,loc112444864,loc101905498,loc104969159,and loc104974530)levels were verified.The results showed that the results of RT-q PCR and RNA-seq sequencing were basically the same,indicating that the results of RNA-seq sequencing were credible;the differentially expressed m RNAs were enriched by GO and KEGG pathways The analysis showed that the differentially expressed m RNAs were significantly enriched in TNF,NLRs,NF-?B,apoptosis,Hippo,TLRs and other signal pathways;the differentially expressed lnc RNAs and m RNAs were analyzed by the lnc RNAs-m RNAs co-expression network analysis,and a total of Five lnc RNA-m RNA co-expression networks;further analysis revealed that lnc RNA loc104974530 and MAPK8,a key factor in regulating apoptosis Among the positive regulatory relations.Secondly,this study explored the effect of lnc RNA loc104974530 on the regulation of MAPK8 transcription on apoptosis and its mechanism.Studies have shown that the expression level of host lnc RNA loc104974530 is significantly reduced after BVDV infection with MDBK,and the m RNA and protein expression levels of MAPK8 are also significantly reduced.At the same time,the transcription levels of apoptosis-related genes Caspase-3 and anti-apoptotic gene Bcl-2 And protein expression levels increased significantly,indicating that BVDV infection promoted the occurrence of apoptosis,and that the infected cells showed early apoptosis p eaks(flow analysis).RNAscope analysis showed that lnc RNA loc104974530 was mainly located in the nucleus and MAPK8 was located in the cytoplasm.When BVDV infected MDBK cells,MAPK8 in the cytoplasm was recruited into the nucleus by lnc RNA loc104974530.In addition,lnc RNA loc104974530 has a regulatory effect on viral replication in the early stages of BVDV infection.Overexpression of lnc RNA loc104974530 increases the virus' s replication capacity within 24-48 hours of viral infection,meanwhile,the m RNA transcription level and protein expression level of MAPK8 also increase,leading to apoptosis.The m RNA transcription level and protein expression level of related genes decreased,which further confirmed that there is a positive regulatory relationship between lnc RNA loc104974530 and MAPK8.Meanwhile,the results of flow cytometry showed that after overexpression of lnc RNA loc104974530,the apoptosis rate decreased and Caspase-3 activity decreased,indicating that overexpression of lnc RNA loc104974530 can inhibit BVDV-induced apoptosis and regulate Bcl-2 and Caspase-3 expression.Our study showed that loc104974530 expression was significantly reduced during BVDV AV69 infection,especially at 48 h.At the same time,the transcription level and protein expression level of MAPK8 decreased significantly.After BVDV AV69 infection,the apoptosis level increased,and the transcription level and protein expression level of apoptosis-related genes Caspase-3 and anti-apoptotic gene Bcl-2 increased significantly.Flow cytometry showed that early apoptotic peaks appeared after BVDV infection,and the cell content in the synthesis phase decreased.Compared with the control group,the apoptosis rate increased after BVDV infection.Through RNAscope experiments,it was confirmed that loc104974530 was localized in the nucleus and cytoplasm of MDBK,and most of them were located in the nucleus.After BVDV infection,MAPK8 was recruited into the nucleus by loc104974530 in the nucleus.loc104974530 has a regulatory effect on virus rep lication in the early stage of BVDV AV69 infection.After overexpression of loc104974530,the virus' replication ability is enhanced within 24-48 h of infection.After over-expression of loc104974530,the expression of loc104974530 increased significantly by RT-q PCR,the expression of MAPK8 also increased simultaneously,and the expression of apoptosis-related genes decreased,confirming the positive regulatory relationship between the two genes.When the expression of loc104974530 increased,Western blot experiments found that the protein expression of MAPK8 increase,and the expression of apoptosis-related proteins will decrease.Flow cytometry detected a decrease in apoptosis and Caspase3 activity after overexpression of loc104974530.It is shown that overexpression of loc104974530 can inhibit BVDV AV69-induced apoptosis and Caspase-3 activity,and regulate the expression of Bcl-2 and Caspase-3.In summary,this study used RNA-seq sequencing to obtain lnc RNA differential expression profiles after BVDV infected host cells MDBK.Through lnc RNAs-m RNAs co-expression network analysis,we screened out lnc RNAs that are potential regulators of key apoptosis factor MAPK8 loc104974530,and studied the effect of lnc RNA loc104974530 on MAPK8 transcription on apoptosis and its mechanism.The results show that host cells can be infected with BVDV in the early stage by inhibiting the expression of lnc RNA loc104974530 to inhibit the m RNA and protein expression of MAPK8,and indirectly Promote the transcription and expression of Bcl-2 and Caspase-3,cause cells to undergo autonomous apoptosis to inhibit virus replication;in the late stage of viral infection,BVDV induces up-regulation of the expression level of lnc RNA loc104974530,and then induces passive cell apoptosis to promote release of virions.The results of this study provide a theoretical basis for further understanding the interaction mechanism between BVDV and host cells during the pathogenesis of BVDV infection.