Expression Of Circular RNA CircKIAA In Bovine Mastitis Tissue And Its Role In Bovine Mammary Epithelial Cells

Bovine mastitis is a disease with high incidence and infection rate in dairy farming.It not only can leads to milk production and milk quality decline,but also reduces milk production,which leads to a huge impact on the development of dairy industry.Studies have showed that circular RNA(circ RNA)can be used as a biomarker and therapeutic target in a variety of inflammatory process,but whether circ RNA can be used as a potential therapeutic target for bovine mastitis is not clear.An Escherichia coli(E.coli)induced mastitis cell model was established in our previous research.A number of differentially expressed circ RNAs were screened by high-throughput sequencing.Bioinformatics analysis showed that one of the circ RNAs was associated with cellular immune response and proliferation.Since KIAA0556 is the genetic marker for this circ RNA,it was named circ KIAA.In this study,the pathological and pathological bacteria in the residual milk were isolated and identified from the breast tissue of the acquired mastitis cows and normal cows.Real-time quantitative PCR was used to detect and compare the normal isolates of circ KIAA and E.coli.The expression level of inflammatory mammary tissue was found to be significantly higher in inflammatory inflammatory mammary tissue than in normal mammary tissue.In the in vitro mastitis cell model induced by E.coli cell wall compoments,lipopolysaccharide(LPS),the expression level of circ KIAA was also higher than that of the normal control group.Then,the mammary gland epithelial cell line with stable over-expression of circ KIAA was established.The circ KIAA was overexpressed to investigate its effects on inflammatory responses,proliferation,and apoptosis by real-time fluorescent quantitative PCR,flow cytometry,CCK-8 cell viability assay and EDU cell proliferation assay.It was found that overexpression of circ KIAA enhanced LPS-induced expression of inflammatory cytokines TNF-a,IL-1β and IL8,inhibited proliferation of mammary epithelial cells and induced apoptosis.The software mi RBase predicts that circ KIAA binds to mi R-326,while mi Randa and Targetscan software predict that Bcl-2 is one of the potential targets for mi R-326.Further studies in the study found that the expression level of Bcl-2 was lower in the overexpressed circ KIAA group than in the control group,but also lower in the cell group co-expressing circ KIAA and mi R-326 than in the overexpressing circ KIAA group,indicating that circ KIAA exerts on mi R The "sponge" effect of-326 regulates the expression of Bcl-2,promotes apoptosis of MAC-T cells and inhibits proliferation of MAC-T cells.In conclusion,expression levels of circKIAA in bovine mammary tissue and epithelial cells under inflammation was significantly increased,thus participated in the process of bovine mastitis.Increased circ KIAA expresssion in mammary tissuse under inflammatory conditions regulates mammary epithelial cell proliferation and apoptosis through the sponge effect of micro RNA-326 and the expression of Bcl-2,The findgins ot this sutdy would provide a new therapeutic target for the prevention and treatment of bovine mastitis.