Unknown,Transcriptomics,Genomics,Proteomics

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A next generation sequencing approach to profiling the host miRNA response to a bovine mastitis pathogen

ABSTRACT: Substantial evidence is now beginning to emerge that miRNAs, short, non-coding RNAs, which post-transcriptionally regulate gene expression, play a key role in the regulation of innate and adaptive immunity in humans and mice. Little is currently known, however, regarding the importance miRNAs in regulating the host response to infection in agriculturally important animals, such as cattle. Mastitis is an inflammatory disease of the mammary gland caused either by infection or physical damage, which is associated with substantial economic losses. In this study, we report a next generation sequencing approach to profile the expression of bovine miRNAs in primary bovine mammary epithelial (BMEs) cells challenged with a bovine mastitis pathogen, Streptococcus uberis (0140J). Computational analysis has been undertaken on 450 million raw sequence reads and revealed that 20% of known bovine miRNAs are expressed in BMEs. Furthermore, 22 miRNAs were found to be differentially expressed over the 6 hour time-course. We have completed miRNA target prediction analysis and found that the target genes of down-regulated miRNAs are enriched for having a role in innate immunity, and pathways associated with target genes of up-regulated miRNA correspond to previously reported mastitis-relevant pathways. In addition, we report 21 potentially novel bovine miRNAs that have not previously been described, two of which have close human orthologs. This study provides new insight into the regulation of miRNAs in the host response to infection at an unprecedented level in any species. 24 miRNAseq libraries were prepared from 3 infected and 3 control replicates at 1, 2, 4 and 6 hours.

ORGANISM(S): Bos taurus

SUBMITTER: Nathan Lawless

PROVIDER: E-GEOD-41278 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Establishment of an in vitro system to explore molecular mechanisms of mastitis susceptibility in cattle by comparative expression profiling of Escherichia coli and Staphylococcus aureus inoculated primary cells sampled from cows with different genetic predisposition for somatic cell score Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows that were selected for high and low mastitis susceptibility by applying a marker assisted selection strategy considering QTL and molecular marker information of a repetitively confirmed QTL for SCS in the telomeric region on BTA18 The cells were cultivated and subsequently inoculated with heat inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 hours the cells were harvested and comparatively analyzed using microarray expression chip technology to identify differences in mRNA expression profiles attributed to cultivation, inoculation or to genetic predisposition. Six heifers inheriting the favorable paternal QTL allele and five heifers inheriting the unfavorable QTL allele were selected by applying a marker assisted selection strategy. All heifers were kept under the same environmental conditions, had no clinical mastitis and did not show any indication of bacterial infection at slaughter. Primary bovine mammary gland epithelial cell cultures were established from cells sampled from the udder parenchyma of each cow. The cells were challenged with heat inactivated Escherichia coli and Staphylococcus aureus or with PBS for control. After 1, 6 and 24 hours the cells were harvested and mRNA expression was comparatively analyzed between time points for each treatment and each paternally inherited SCS-BTA18-QTL allele, respectively. In addition, the differences in gene expression at time points 1, 6 and 24h between inoculated and respective uninoculated control cells were investigated using the short time series expression miner STEM for co-expression profiling and GO cattegory enrichment analyses.