Screening And Characterization Of Exosomal MiRNAs And LncRNAs As Biomarkers In Bovine Mastitis

Bovine mastitis is a common and frequently cause of economic losses to the dairy industry.It not only leads to huge economic losses,but also causes serious problems in cows and humans.Staphylococcus aureus causes one of the most common types of mastitis,which is very difficult to be eradicated from mammary glands.This thesis research investigated functions of miRNA and lncRNA in bovine mammary glands and mastitis.The results for these studies were shown as follows:1.The goal of our study was to comprehensively survey by RNA sequencing technology(RNA-seq)the miRNA content of uninfected milk exosomes and S.aureus infected milk exosome,including three normal groups and six S.aureus infected group.Sequencing provided a total of 101,392,712 reads,with an average of 11,265,856 reads for each sample.After removing the low quality reads,adaptors,and insufficient tags and sequences,a total of 94,187,107 clean reads were obtained,with an average of 10,465,234 reads for each sample.A total of 372 new miRNAswasdiscovered.Differentiation analysis demonstrated that 37 mi RNA consisted of 22 known miRNAs and 15 novel miRNAs.bta-miR-1 and bta-mi R-122 were up-regulated 3.79 fold and 16.4 fold,respectively.A total of 10,577 unique genes were predicted by miRanda be targeted based by the 22 DE miRNAs.GO and KEGG functional annotation indicate that the differentially expressed miRNAs participate in the physiological processes of the cells and the inflammatory pathways.2.The goal of our study was to comprehensively survey by RNA sequencing technology(RNA-seq)the lncRNA content of uninfected milk exosomes and S.aureus infected milk exosome,including three normal groups and six S.aureus infected group.Sequencing provided a total of 874,840,266 reads,with an average of 97,204,474 reads for each sample.After removing the low quality reads,adaptors,and insufficient tags and sequences,a total of 845,687,262 clean reads were obtained,with an average of 93,965,251 reads for each sample.A total of 500 lncRNAwas predicted by using the combination of 4 software programs(CPAT,CNCI,CPC and hmmscan).Differentiation analysis demonstrated that 9 lncRNA consisted of 4 up-expressed lncRNAs and 5 down-expressed lncRNAs.3.The expression of bta-miR-21-3p in MAC-T cells challenged with and without heat-inactivated S.aureus at 30 min,1,2,6,and 12 hour was down-regulated.The target gene of bta-miR-21-3p was identified by double luciferase reporter assay and Western blotting.IL-25 could promote the expression of inflammatory cytokines and Th2 cytokine response.S.aureus elicits a delayed inflammatory response and did not significantly induce proinflammatory cytokines.4.In order to investigate the distribution and function of lincRNA in cow mammary gland,we used a bioinformatics analysis in this study.Using computational methods,184 lincRNAs were identified from 5 RNA-seq datasets from bovine mammary glands.Their non-coding potentials were predicted by using the combination of 4 software programs(CPAT,CNCI,CPC and hmmscan).By comparing locations of our 184 lincRNAs with QTL positions,113 different lincRNAs were located in 399 cattle QTLs.For example,one lincRNA was located in clinical mastitis QTLs and 36 lincRNAs in 169 unique milk related QTLs.5.In order to find potential distributions of ACME element in livestock-associated staphylococci,a total of 146 staphylococcal isolates from three dairy farms in Northwestern China were screened.Among the 146 staphylococcus species,34 strains were S.haemolyticus,37 strains were S.aureus,24 strains were S.epidermidis,19 strains were S.chromogenes,2 strains were S.agnetis and 30 strains were S.warneri.The full length of ACME-SCCmec composite island of Y24 was determined.This is the first report on the existence of ACME in livestock-associated staphylococci.Our recent studies also suggested occurrence of horizontal gene transfer between staphylococci in bovine and staphylococci in human.Wide existence of ACME in livestock-associated staphylococci from this study and a potential risk of spreading ACME among different staphylococcal species warrant close monitoring and further studies.Above all,our study of miRNA and lncRNA expression from uninfected milk exosomes and S.aureus infected milk exosome provides new and comprehensive information supporting a role of some specific miRNA or lncRNA involved in host immune response.Identification of different expressed exosome miRNA and lncRNA expanded the repertoire of miRNA and lncRNAs for dairy cattle and consequently will help understand functions of the bovine mi RNA and lncRNAs in bovine genome.It can also serve as potential molecular markers to detect mastitis.In addition,this is the first report on the existence of ACME in livestock-associated staphylococci.