Study On MiRNA Sequencing And Analysis Of Exosomes From MDBK Infected With BVDV

Bovine viral diarrhea virus(BVDV)can infect cattle,pigs,sheep,deer and other livestock,causing fever,diarrhea,decreasing productivity,reducing milk yield and other symptoms in infected animals,especially cattle.It has high incidence rate and mortality,causing great harm to the global cattle industry.Exosome is a kind of extracellular vesicle produced by mammalian cells for intracellular communication.The nucleic acid,protein and virions of various envelope RNA viruses can be wrapped by exosomes to achieve intercellular transmission.Studies have shown that BVDV can promote the production of exosomes and thus promote their own replication.As a single stranded RNA molecule with regulatory function,miRNA is also one of the important components of exosomes.At present,there are few studies on exosomes produced by BVDV-infected cells,and even fewer further studies on miRNA produced by BVDV-infected cells in exosomes.Therefore,if we can analyze the miRNA produced by BVDV-infected cells in exosomes,then predict and analyze the function of corresponding target genes,this study will provide new theoretical data to elucidate the molecular mechanism of BVDV influencing host cells to assist virus infection through exosomes.Bovine kidney cells(MDBK)were cultured with serum substitute instead of FBS and serum-free.The serum-free MDBK was inoculated with BVDV virus.After 72 hours,the cells were freeze-thawed repeatedly to collect BVDV virus.The standard curve of BVDV was established by fluorescence quantitative PCR,and the efficiency of BVDV infection with MDBK was determined.MDBK was inoculated with a high concentration of BVDV virus.The cells were collected 72 hours later,and MDBK exosomes were collected by supercentrifugation.The morphological changes of vesicles of MDBK exosomes infected with BVDV and those without BVDV were observed by electron microscopy.Subsequently,Western blot was used to detect the expression of exosome marker proteins CD63 and ALIX.The results showed that both MDBK exosomes infected with BVDV and those not infected with BVDV expressed CD63 and ALIX proteins,but did not express cytoplasmic marker protein Calnexin.This indicates that we have successfully obtained MDBK exosomes infected/uninfected with BVDV,which can be used for subsequent experiments.Total RNA of MDBK exosomes infected/uninfected with BVDV was extracted by Trizol method,and small RNA libraries of MDBK exosomes infected/uninfected with BVDV were constructed.The quality of each small RNA library was evaluated by Nanodrop spectrophotometer,Qubit fluorescence quantification and Agarose gel electrophoresis.The results showed that the RNA in the 6 small libraries were highly purified and free from protein and genome contamination,which could be sequenced and analyzed.Subsequently,the quality of sequencing results was evaluated by Q30,and the results showed that the Q30 of each RNA library was about 98%,indicating that the transcriptome high-throughput sequencing results were good and could be used for further analysis.The sequences were compared with miRbase database,miRDeep2 software and RNAfold software,and a total of 678 miRNAs were detected,including 421 known miRNAs and 257 novel miRNAs.Then,the number of tags on each mature miRNA was statistically compared as the original expression quantity of the miRNA,and the expression quantity of miRNA was standardized by TPM.The results showed that a total of 71 highly expressed miRNAs were detected after BVDV infection,including 4 novel miRNAs.By comparing P-value and Fold Change of MDBK exosome samples infected/uninfected with BVDV,differentially expressed miRNAs were screened.The results showed that the expression levels of 126 miRNAs were significantly changed after BVDV infection compared with those of MDBK exosome miRNAs not infected with BVDV.There were 84 up-regulated miRNAs and 42 down-regulated miRNAs.Cluster analysis of differentially expressed miRNAs using Heatmap2 function of R packet showed that there were clustering phenomena in the exosome of MDBK infected with BVDV and without BVDV.In order to further study the molecular mechanisms related to the influence of BVDV infection on host cells through exosomes and its aids to virus infection,we used Targetscan website and miRanda website to predict the potential target genes of the top 5 miRNAs with significant differences.Then we obtained the final target genes by intersection of Venn diagram.The results showed that 47 target genes were predicted by significantly up-regulated miRNA and 30 target genes were predicted by significantly down-regulated miRNA.Targetscan was used to analyze the target relationship between miRNA and target genes,and Cytoscape software was used to draw the miRNA-target genes network diagram,and the results showed that IFNAR1 gene is co-regulated by bta-miR-novel-chr2＿2377,bta-miR-novel-chr3＿3939 and btamiR-7.SIK1 gene is co-regulated by bta-miR-novel-chr2＿2377 and bta-miR-Novelchr3＿3939,bta-miR-16 a and bta-miR-6524.Then,software GO Category online analysis was used for functional enrichment analysis of target genes,and the results showed that the target genes of the top 5 miRNAs with significant differences were mainly enriched in GO items such as type I interferon signaling pathway,cytokine activity,immune receptor activity and plasma membrane composition.Finally,KEGG pathway analysis was conducted on target genes differentially expressed miRNA in MDBK exosome after infection with BVDV.The results showed that the top 5miRNA target genes with significant difference were mainly enriched in folate resistance,Hepatitis C,JAK-STAT signaling pathway,Toll-like receptor signaling pathway and other signaling pathways.The above results indicate that BVDV infection affects the expression pattern of miRNA in the exosomes of MDBK cells,and the target genes of differentially expressed miRNA are mainly enriched in type I interferon signaling pathway,cytokine activity and JAK-STAT signaling pathway.This study provides new theoretical data to elucidate the molecular mechanisms by which BVDV influences host cells through exosomes.