Screening Of MicroRNAs In Immune Response To S.aureus Isolated From Bovine Mastitis And Their Biological Functions

Bovine mastitis is an inflammatory response of the mammary tissues of cows stimulated by the external environment,mainly due to the infection of various pathogenic microorganisms,and S.aureus is one of the most common pathogens of mastitis in cows,which causes subclinical and chronic clinical forms of mastitis in cows that are difficult to diagnose and treat.Studies have shown that early interactions between S.aureus and bovine mammary epithelial cells(bMECs)trigger both the innate and acquired immune systems in bovine mammary tissue.However,microRNA(miRNA)has been shown to have a role as a key target molecule in the intrinsic immune response of the dairy mammary gland,resistance to pathogenic bacterial infection,and immune regulation of inflammation,and can be used as a potential biomarker for the diagnosis and treatment of mastitis in dairy cows.Therefore,there is an urgent need to explore how miRNAs regulate the innate defense mechanisms of the dairy mammary gland against invading pathogens in the early stages of infection in order to propose appropriate strategies for the control of Staphylococcus aureus mastitis in dairy cows.In this study,bMECs were stimulated with Staphylococcus aureus,and differentially expressed miRNAs in bMECs were screened by Solexa high-throughput sequencing;stable real-time fluorescent quantitative PCR assay of bta-miR-29b was established by RT-qPCR;potential candidate miRNAs were used to study the biological functions of bta-miR-29b in bMECs by gene overexpression or repression methods.This project aims to reveal the regulatory role and molecular mechanism of miRNAs in the immune response of bMECs against S.aureus,and provide a theoretical basis for elucidating the pathogenesis of S.aureus mastitis and screening candidate molecular markers for early diagnosis.The experimental results showed that 38710696 Unique reads were obtained by Solexa highthroughput sequencing,of which 10826771 were Valid reads,accounting for 28.21%of the total data;the Unique reads were 18-26 nt in length,with a major peak of 22 nt.The sequencing data were compared with the miRBase database,and 3442 Unique miRNAs were identified,of which 1625 were known miRNAs and 1817 were novel miRNAs.The sequencing data were compared with the miRBase database,and 3442 Unique miRNAs were identified,including 1625 known miRNAs and 1817 novel miRNAs.Among the 174 differentially expressed miRNAs,122 miRNAs were up-regulated and 52 miRNAs were down-regulated.Using TargetScan and miRanda software,6476 target genes were predicted.GO enrichment analysis showed that the target genes were mainly enriched in signal transduction regulation,immune response,protein transport across membranes,cell differentiation and bioadhesion;KEGG functional annotation analysis showed that the target genes were mainly enriched in immune-related pathways including human immunodeficiency virus type 1 infection and Barr virus infection pathways,interleukin-17 signalling and chemokine signalling pathways.The results of the functional analysis of the target genes of miRNAs indicate that the candidate molecule bta-miR-29b has potential research value in the defense against pathogenic microbial infections.Using the potential candidate bta-miR-29b as the target,we established a stable real-time fluorescent quantitative PCR(RT-qPCR)assay.The method is non-cross-reactive and specific with bovine Escherichia coli and Streptococcus aureus;the minimum cDNA template amount detection concentration is 1×10-4 pg/20 μL,which is approximately 100-fold more sensitive than conventional PCR;the coefficients of variation of the reproducibility tests are all less than 1%,indicating that the method is reproducible.The established RT-qPCR method was applied to detect the expression of bta-miR-29b after S.aureus was infected with bMECs,and the expression of bta-miR-29b was found to be highly significantly down-regulated(P<0.01),which was consistent with the results of Solexa high-throughput sequencing and proved that the method was accurate and reliable.The biological functions of bta-miR29b in bMECs were investigated using gene overexpression and repression methods.The results showed that overexpression of bta-miR-29b altered the morphology of mammary epithelial cells in dairy cows;overexpression of bta-miR-29b mimic significantly inhibited the growth viability of bMECs(P<0.01)and also significantly inhibited the proliferative capacity of bMECs(P<0.05),while inhibition of bta-miR29b significantly promoted the growth viability of bMECs(P<0.05)and significantly increased the proliferative capacity of bMECs(P<0.05).Meanwhile,overexpression of bta-miR-29b resulted in significant down-regulation of TNF-α,IL-1β,IL-6 and IFN-γ secretion(P<0.05),and inhibition of btamiR-29b caused significant up-regulation of IL-1β and IL-6 secretion(P<0.05).The experimental results suggest that this will lay the theoretical foundation for the subsequent selection of bta-miR-29b as a candidate molecular marker for the early diagnosis of Staphylococcus aureus mastitis in dairy cows.