Study On Construction Of A Small RNA Library Of BVDV Infected Cells And The Regulation Of Apoptosis And Autophagy By MiR-2904

Bovine viral diarrhea is a contagious disease caused by bovine viral diarrhea virus,mainly causes bovine fever,diarrhea,leukopenia,immunosuppression,mucosal hyperemia and other clinical symptoms,but also causes the cow milk yield decline,pregnant cow abortion,stillbirth and persistent infection in cattle.The key factors influencing BVDV persistent infection prevention and control is the lack of in-depth understanding of the major basic theory problems of BVDV persistent infection formation and maintenance mechanism.The key to the formation and maintenance of BVDV persistent infection involves two levels of target cells and organism.A large number of studies show that micro RNA plays an important role in the interaction between pathogen and host cells.Therefore,in this study,after the cells was infected by virus,we will focus on the miRNA regulation of cell apoptosis,autophagy and immune to reveal BVDV mechanism of persistent infection.Objective: To investigate the molecular mechanism of BVDV persistent infection by the small RNA molecules on apoptosis,autophagy and BVDV replication in BVDV infected cells.Methods:(1)The total RNA of normal MDBK cells(MDBKCs)and cpBVDV NADL infected cells(BMDBKCs)was extracted and used to construct the miRNA and mRNA libraries.The data was analyzed and verified by the bioinformatics technologies software;(2)The miR-2904 oligonucleotide was transfected into MDBK cells to detect cell apoptosis rate by inverted microscope,hoechst staining and flow cytometry respectively;Caspase-9,the target gene of miR-2904 in apoptosis pathway was predicted and identified;Caspase-9 mRNA and protein expression levels were detected in MDBK cells transfected with miR-2904 oligonucleotides;(3)MDBK cells were infected with cpBVDV NADL and the autophagy was detected by transmission electron microscopy;The GFP-LC3 plasmid was transfected into MDBK cells and the GFP-LC3 plasmid and 2904 mimics/inhibitor/NC mimics were co-transfected into MDBK cells,the level of autophagy was detected by laser scanning confocal microscopy,respectively;ATG13,the target gene of miR-2904 in autophagy pathway was predicted and identified;ATG13 mRNA and protein expression levels were detected in MDBK cells transfected with miR-2904 oligonucleotides;(4)IL-6?IL-1 beta and IFN-a mRNA levels were detected after MDBK cells infected with cpBVDV NADL;IL6R,the target gene of miR-2904 in immune pathway was predicted and identified;Detection of IL6 R mRNA and protein expression and cpBVDV NADL replication in MDBK cells transfected with miR-2904 oligonucleotides;Results:(1)290,280,287 and 298 conservative mi RNA and 1042,231,354 and 775 new miRNA were identified in the control group(CK),ck-vs-2 h?ck-vs-6 and ck-vs-18 h respectively by small RNA sequencing;283,84 and 54 significant difference miRNA were identified in ck-vs-2h,ck-vs-6h and ck-vs-18 h,respectively,in which there were 18 differentially expressed miRNAs were expressed in different time;The results of high-throughput sequencing were almost consistent with the results of real-time quantitative PCR;By RNA-seq sequencing,232 differentially expressed genes were identified in ck-vs-2h,135 genes differentially expressed were identified in ck-vs-6h,185 genes differentially expressed were identified in ck-vs-18 h,in which the were 43 differentially expressed genes were expressed in different time;The replication of cpBVDV NADLwas inhibited by miR-2904;The predicted target genes of apoptosis,autophagy and immunity of miR-2904 were 11,6 and 34,respectively;(2)The apoptosis rate of MDBK cells was decreased by miR-2904;miR-2904 can target caspase-9 3' UTR directly and inhibit the expression of caspase-9;(3)The number of autophagy or autophagic lysosome was increased by cpBVDV NADL infection;The GFP-LC3 fusion protein was presented in the cells transfected with GFP-LC3 plasmid;The number of GFP-LC3 positive cells was significantly reduced by co-transfection of GFP-LC3 and miR-2904 mimics;miR-2904 can target ATG13 3'UTR directly and inhibit its expression;(4)The levels of IL-6?IL-1 beta and IFN-a mRNA were increased after cpBVDV NADL infecting;miR-2904 can target IL6 R 3' UTR directly and inhibit its expression;Over expression of miR-2904 can reduce cpBVDV NADL replication level.Conclusion: miRNA and mRNA libraries of BVDV infected cells were successfully constructed,and a group of differentially expressed miRNAs was obtained.,miR-2904 could significantly inhibit the replication of BVDV,the possible mechanism of viral replication is that mi R-2904 inhibits cell apoptosis and autophagy by targeting target genes,thereby affecting the replication of BVDV.