Expression Of Circular RNA CircLPP In Bovine Mastitic Tissues And Its Application As A Potential Therapeutic Target

Bovine mastitis is one of the most common diseases in dairy farming.It is a major factor to restrict the healthy development of dairy farming and leads to huge economic losses to the dairy farming industry.Except for strengthening managements of feeding,attaching importance to environmental sanitation and milking hygiene,a lot of treatments,including antibiotics,Chinese herbal medicines,biological agents and biologically active extracts have been used for the prevention and treatment of bovine mastitis.But the prevention and control work are still very serious.In recent years,studies have demonstrated that the expression and role of circular RNA(circRNA)play a role in the development of various diseases.The fact of its differential expression in normal and disease-related samples and the gradual clarification of circRNA functions in diseases make circRNA possible to be new diagnostic markers for clinical diseases and have potentials to be effective therapeutic targets.In this study,high-throughput sequencing analysis of normal and inflammatory bovine mammary epithelial cells from cows was performed to obtain differentially expressed circRNAs.A newly discovered circRNA was selected and named circLPP,and its expression in normal mammary tissue and inflammatory properties in dairy cows were studied.The increased expression of circLPP in mammary tissue under inflammatory conditions on inflammatory cytokine secretion,viability and proliferation was studied to clarify whether the circRNA is involved in the process of bovine mastitis.CircLPP is expected to become a new target for the treatment of bovine mastitis and a new gene for the bovine breeding resistant to mastitis.Following results were obtained.1.Bioinformatics analysis of high-throughput sequencing data revealed that circLPP was derived from the first exon of the parental gene LIM domain containing preferred translocation partner in lipoma(LPP),so it was named circLPP.The predicted target microRNA of circLPP was miR-615,and SPRED3 is the target mRNA downstream of miR-615.The results of real-time quantitative PCR showed that the expression level of circLPP in the mammary gland of bovine mastitis was significantly higher than that in the normal mammary tissue,indicating that circLPP is involved in the process of bovine mastitis.2.Bovine mammary epithelial cells of MAC-T with circLPP overexpression wasestablished by stably overexpressing circLPP.Through a series of experimental methods,it was found that overexpression of circLPP can induce the secretion of inflammatory cytokines TNF-?,IL1-? and IL-6 in bovine mammary gland epithelial cells;decrease the viability of mammary epithelial cells and inhibit the proliferation,indicating increased expression of circLPP in inflammatory states has an important influence on the biological functions of mammary epithelial cells including inflammatory response and proliferation.3.The upstream and downstream regulation of circLPP?miR-615?SPRED3 was clarified by plasmid transfection,using miR-615 mimics and inhibitors,and it was found that circLPP functions via the axis of circLPP?miR615?SPRED3.These functions include to regulate the inflammatory response and proliferation of mammary epithelial cells and then participates in the development and progression of mastitis in dairy cows.In summary,the results of this study indicate that the expression level of circLPP is significantly increased in mammary tissue under an inflammatory condition,which plays an important role in regulating the inflammatory response,proliferation and viability of mammary gland epithelium,thus involved in the inflammation process of the bovine mammary glands.The findings of this study suggest that circLPP may be a target for future prevention and treatment of mastitis in dairy cows,and a candidate gene for bovine breeding resistant to mastitis.