Expression And Mechanism Of CircRNA IL27RA In Mastitis Of Dairy Cows Induced By LPS

The mammary gland safety directly of dairy cows affects milk production and restricts the dairy economy.Dairy cow mastitis is one of the main diseases in dairy cow production.Many factors cause mastitis in dairy cows,pathogenic bacteria account for the vast maj ority,and the most common one is Escherichia coli.When Escherichia coli invades the udder of dairy cows,the antibody on the cell membrane surface will specifically recognize the unique component of the Gram-negative bacteria-lipopolysaccharide（LPS）cell wall.Then activate the cellular immune response,trigger an inflammatory response,and endangers the life safety of dairy cows in severe cases.Circular RNA（circRNA）is a newly discovered endogenous non-coding RNA.Many studies have shown that circRNAs play different biological functions in different parts of different species.The research on circRNA in livestock started late.In recent years,reports on bovine circRNA have gradually increased,but they mainly focus on muscle growth and development,and more research needs to be done on cow mastitis.In this study,high-throughput sequencing was carried out using the mammary gland inflammatory tissue infected with E.coli and normal breast tissue provided by the laboratory as controls.The results showed that 10,437 circRNAs were identified,the main distribution length was 200-800 bp,and the average length was 2,981 bp.Sixty-four significantly differentially expressed circRNAs were selected by bioinformatics analysis,of which 46 were up-regulated,and 18 were down-regulated.KEGG and GO enrichment analysis was performed on the parental genes of differential circRNAs.We selected circRNA₀3225 located in the most significantly enriched Th17 cell differentiation pathway as the research object,and its mother gene is Interleukin 27 receptor subunit alpha（IL27RA）.It was named circIL27RA according to its parental gene.Experiments showed that circIL27RA was formed by connecting the first positions of exons 5 and 6 of the IL27RA gene,and the amplified length was 234 bp.After functional prediction and expression trend verification,it was confirmed that circIL27RA was involved in the cellular immune response.Using LPS as an inflammatory endotoxin,we established a mammary epithelial cell inflammation model for the functional study of circIL27RA through Cell Counting Kit-8（CCK-8）,Flow Cytometry（FCM）,and Real-time quantitative polymerase chain reaction（RTqPCR）experiments.First,the CCK-8 experiment showed that the percentage of cell survival was lower when the treatment time was 6 hours.We used flow cytometry to detect the apoptosis of cells treated for 6 h and added with different doses of LPS.The results of FCM showed that the early apoptosis rate of cells was the highest when the adjusted concentration of LPS was 100 ng/mL.Subsequent RT-qPCR detection results showed that the expressions of IL6,IL8,TLR4,and NF-κB were extremely significantly up-regulated.Therefore,it was finally determined that the concentration of LPS to construct the inflammation model of dairy cow mammary gland epithelial cells was 100 ng/mL,and the treatment time was 6 hours.In order to explore the function of circIL27RA,we performed subcellular localization of circIL27RA by nuclear-cytoplasmic separation technique.We found that circIL27RA is mainly present in the cytoplasm.EdU,FCM,and RT-qPCR experiments showed that overexpression of circIL27RA can inhibit cell apoptosis,promote cell proliferation,activate TLR4/NF-κB signaling pathway in the state of cell inflammation,and enhance the production of IL6 and IL8 pro-inflammatory factors.Interfering with circIL27RA expression increased cell apoptosis and inhibited cell proliferation.RT-qPCR results showed that the expressions of IL6,IL8,TLR4,and NF-κB were extremely significantly down-regulated.Furthermore,the experiment also found that circIL27RA has a favorable regulatory tendency to its parental gene.Therefore,this study conducted a functional study of the circIL27RA maternal gene.We performed IL27RA overexpression and interference treatment in mammary epithelial cells.EdU,FCM,and RT-qPCR experiments showed that IL27RA could significantly inhibit the apoptosis of mammary epithelial cells under inflammation,promote cell proliferation,activate TLR4/NF-κB signaling pathway,and enhance the production of IL6 and IL8 pro-inflammatory factors.In summary,we identified 64 differential circRNAs through high-throughput sequencing of inflammatory mammary gland tissues and normal tissues.circIL27RA located in the most significantly different pathway was selected as the research object.The mammary epithelial cell inflammation model was successfully established by adding LPS to normal mammary epithelial cells to adjust the concentration to 100 ng/mL,and the treatment time was 6 hours.circIL27RA positively regulates the parental gene IL27RA.Overexpression of circIL27RA and IL27RA can inhibit cell apoptosis,promote cell proliferation,activate the TLR4/NF-κB signaling pathway in the state of cell inflammation,and enhance the production of IL6 and pro-inflammatory factors,thereby helping cells Participate in the immune response.