Study On Construction Of LncRNA Library Of BVDV Infected MDBK Cells And The Regulation Of Apoptosis By LncRNA 1620

Bovine viral diarrhea virus（BVDV）is a serious global pathogen of bovine infectious diseases,causing huge economic losses to the cattle industry.At present,there is no effective prevention and control method.One of the main reasons is the persistent infection caused by BVDV.Long non-coding RNA（lnc RNA）has attracted researchers’ attention in recent years because of its important role in regulation of transcription.Previous studies have confirmed that lnc RNA participates in a series of interactions between virus and host,including innate immune response in which lnc RNA plays an important regulatory role.Studies have shown that apoptosis is crucial to the innate immune response caused by virus infection,and virus-induced apoptosis can inhibit or promote virus replication and transmission.Therefore,this study constructs differential lnc RNAs and m RNAs expression profiles of different biotypes BVDV infected MDBK cells through high-through sequencing,to analyse the cellular pathways and functions of these differential expression genes,to screen lnc RNAs that regulates cell apoptosis and affects virus replication during BVDV infection,and to study the pathway and internal mechanism of lnc RNAs regulating cell apoptosis.The study explored the interaction between BVDV and host cells from the perspective of lnc RNAs,and screen out the functional lnc RNAs.Studying the mechanism of BVDV inducing cell apoptosis and affecting BVDV replication is a new exploration of BVDV prevention and treatment,providing a new perspective for revealing the pathogenic mechanism of BVDV and laying a foundation for studying the persistent infection mechanism of BVDV.Objective:（1）Construction of lnc RNA and m RNA library of cp or ncp BVDV infected MDBK cells;（2）To analyze the pathway of differentially expressed genes induced by two biotypes viruses and the cell processes participate in,and to provide a new scientific basis for the study of the mechanism of persistent infection and BVDV pathogenesis in cell level;（3）Screening out the apoptosis-related lnc RNA during BVDV infection,to study its function of regulating cell apoptosis and virus replication;（4）Futher study the mechanism of action of lnc RNA,providing new point of views for the prevention and control of BVDV,and providing reference for the study of the lnc RNA functions of bovine in disease process.Methods:（1）BVDV NADL（cp biotype）and BVDV C17（ncp biotype）infected MDBK cells 2h,6h,18 h,TCID50,q RT-PCR and cell immunofluorescence were used to determine the dose of BVDV NADL and BVDV C17 infection.7 total RNA samples were extracted: control,Cp₂h,Cp₆h,Cp₁8h,Ncp₂h,Ncp₆h,and Ncp₁8h.Construction of RNA-seq library for qualified RNA samples and sequenced in the Illumina Hiseq 4000 sequencing platform.The raw data is filtered to obtain high-quality reads.（2）Reference genome comparison analysis,expression level analysis,genome characters analysis and alternative splicing analysis were carried out by bioinformatics methods.Screening of known lnc RNA transcripts and newly predicted lnc RNA transcripts.Adjusted P-values﹤0.05 and log2（fold change）≥1 were set as the cut-off values for differentially expressed genes（DEGs）.GO enrichment and KEGG pathways analysis were proformed based on DEGs.Significantly DEGs were randomly selected to validate the results of q RT-PCR were consistent with high-throughput sequencing.（3）Through bioinformatics analysis,screening out the apoptosis-related lnc RNA ALDBBTAG0000001620,abbreviated as lnc RNA 1620.The localization of lnc RNA 1620 in MDBK cells was detected by fluorescence in situ hybridization.Through the methods of si RNA interference,q RT-PCR,flow cytometry and Caspase-3 activity analysis,to detect the effect of lnc RNA on MDBK cell apoptosis,apoptosis-related genes and virus replication during BVDV infection.（4）Through the methods of si RNA interference,q RT-PCR and Western blot,to detect the effect of lnc RNA1260 on BIRC3 transcription and protein expression level during BVDV infection.The effect of target gene BIRC3 on lnc RNA 1620 transcription was detected by interfering target gene BIRC3 and q RT-PCR.The regulatory effects of lnc RNA 1620 on target gene BIRC3 promoter were detected by double luciferase reporter gene technique.The Ch IRP-q PCR method was used to verify the regulation of lnc RNA 1620 to the DNA level of the target gene BIRC3.Results:（1）MDBK cells infected with cp or ncp BVDV at 100 TCID50/0.1m L.The quality of RNA samples is qualified for RNA-Seq.77.71 GB of clean reads were obtained for subsequent analysis.（2）The sequence of each sample mapping to reference genome of bovine was more than 80%;the expression level of each sample was similar,except for Cp₁8h was higher than others;the expression level and conservation of lnc RNAs were lower than m RNAs,the transcript length and open reading frame length of lnc RNAs were shorter than m RNAs;the exon number of lnc RNAs was fewer than m RNAs;the various types of alternative splicing events in each sample are similar;a total of 7250 newly predicted lnc RNA transcripts and 333 known lnc RNA transcripts were obtained.A total of 5053 DEGs in BVDV NADL and BVDV C17 infection of all stages.The GO and KEGG analysis of differentially expressed lnc RNAs target genes showed that cp or ncp BVDV infection groups were enriched in apoptosis,innate immunity,inflammatory reaction,autophagy and other related pathways,as well as molecular metabolism and other biological processes.The results of q RT-PCR detection of DEGs showed that the differential expression of most genes was consistent with that of RNA-Seq.（3）lnc RNA 1620 was expressed in both nucleus and cytoplasm,and most of them were located in that nucleus.After BVDV NADL infected MDBK cells,the expression of lnc RNA 1620 was significantly up-regulated,and the expression of apoptosis-related genes Bcl-2 and Caspase-3 was also significantly up-regulated at the early stage of virus infection.Caspase-3 significantly down-regulated after interference with lnc RNA interfering with lnc RNA 1620 and infected with BVDV NADL 12 h and 24 h significantly up-regulated the expression of Caspase-3 and Bcl-2.Interference with lnc RNA 1620 significantly promoted the transcription of BVDV NADL 5’ UTR in early infection stage.Interference with lnc RNA 1620 could inhibit BVDV induced apoptosis.The activity of Caspase-3 decreased significantly after interfering with lnc RNA 1620 and infected with BVDV 12 h.（4）After BVDV infected MDBK cells,the transcription level and protein expression level of BIRC3 significantly increased.Interference with the expression of lnc RNA 1620 significantly reduced the transcription level of BIRC3.The changes of transcription levels of BIRC3 and lnc RNA 1620 were shown consistent when MDBK cells were treated with interference of lnc RNA 1620 and BVDV NADL.After interference with the expression of target gene BIRC3,there was no significant change in the transcription level of lnc RNA 1620.The relative luciferase activity increased significantly after co-transfection of BIRC3-p GL4.1 and lnc RNA 1620-pc DNA3.1 plasmid.Ch IRP-q PCR enriched the target gene BIRC3.Conclusion:（1）The RNA-Seq library of cp and ncp BVDV infected MDBK cells was successfully constructed.Clean reads were obtained through data filtering.（2）The reliability of sequencing results was further demonstrated by bioinformatics analysis and q RT-PCR verification such as comparison of bovine reference genome and genome feature analysis.Both cp and ncp BVDV infection of MDBK cells caused the expression of lnc RNAs changes.The cellular pathways and biological processes involved in differential expression of lnc RNAs were different in MDBK cells infected with two biotypes of BVDV,involved in innate immunity,apoptosis,inflammatory reaction,autophagy,molecular metabolism and so on,this study laid a foundation for the research of persistent infection mechanism of BVDV.（3）lnc RNA 1620 expression increased significantly during BVDV NADL infection of MDBK cells,and was involved in regulating cell apoptosis and virus replication.（4）lnc RNA 1620 has a co-expression relationship with target gene BIRC3,lnc RNA 1620 regulates the expression of target gene BIRC3 by enhancing the promoter activity of BIRC3,the molecular mechanism of lnc RNA 1620 regulating cell apoptosis was clarified.