Effects Of CircRNA PRIM2 On Biological Functions And Inflammatory Response Of Bovine Mammary Epithelial Cells

Bovine mastitis is an inflammatory reaction of mammary tissue caused by physical,chemical and microbial infections.The most common microorganisms are Escherichia coli and Staphylococcus aureus.When these two pathogenic microorganisms invade the mammary gland tissue of bovine,lipopolysaccharide(LPS)and lipoteichoic acid(LTA)are used as the main virulence components to stimulate the mammary tissue to produce different degrees of inflammation and even affect lactation.Circular RNA(circular RNA,circ RNA)is a non-coding RNA that has been widely studied in recent years.It is a closed circular RNA molecule produced during the pre-m RNA alternative splicing process.A large number of studies have shown that circular RNA is involved in the regulation of various inflammatory diseases processes,but the relevant role of circ RNA in bovine mastitis is rarely studied.A novel circ RNA was found through high-throughput sequencing analysis in our pervious research,and its expression level was significantly reduced in bovine mammary epithelial cells under an induction of pathogens.In view that this circ RNA is formed by back-splicing of exon 8 and 9 of DNA primase subunit 2(PRIM2),it is named circ PRIM2.In this study,the basic characteristics of the circular RNA were analyzed,following the verification of sequencing results.In addition,effecs of PRIM2 on the proliferation,casein secretion and inflammatory response of bovine mammary epithelial cells were investigated,by overexpressing circ PRIM2 in the mammary epithelial cells.First,a mammary epithelial cells model of bovine mastitis was established by LPS and LTA induction.The expression level of circ PRIM2 was detected by real-time quantitative polymerase chain reaction(RT-q PCR).The expression levels in mammary epithelial cells were significantly reduced,consistent with the results of high-throughput sequencing.Secondly,RT-q PCR was used to detect the expression of circ PRIM2 in different tissues of organs,including breast,heart,liver,spleen,stomach,lung,kidney and intestine of the bovine.Results showed that the expression level of circ PRIM2 was relatively high in lung tissues,while the basic expression level was low in bovine tissues.Furthermore,in order to investigate effects of circ PRIM2 on the functions of mammary epithelial cells,a stably circ PRIM2 overexpressing cell line was established,and the secretion of β-casein was detected by enzyme-linked immunosorbent assay(ELISA).Results showed that overexpression of circ PRIM2 promoted β-casein secretion.The effects of overexpression of circ PRIM2 on the proliferation and vitality of bovine mammary epithelial cells was detected by Ed U method and CCK-8 method.The results showed that overexpression of circ PRIM2 could inhibited the proliferation and vitality of bovine mammary epithelial cells.RT-q PCR was used to detect the effect of its parental gene m RNA level,and it was found that overexpression of circ PRIM2 promoted the expression of its parental gene in inflammatory mammary epithelial cells.Finally,the effect of circ PRIM2 on the inflammatory response of mammary epithelial cells and its preliminary mechanism were explored.The levels of inflammatory cytokines TNF-α,IL-1β,IL-6 m RNA and protein secretion in inflammatory mammary epithelial cell were detected by RT-q PCR and ELISA,respectively.The results showed that overexpression of circ PRIM2 inhibited the m RNA and protein secretion of TNF-α,IL-1β and IL-6 induced by LPS and LTA;western blot was used to detect the expression levels of phosphorylated P65 and total P65 of key signal molecules in the NF-κB signaling pathway.The results showed that overexpression of circ PRIM2 inhibited activation of NF-κB signal pathway by inhibiting phosphorylation P65 activation.In summary,the expression level of circ PRIM2 in mammary epithelial cells was downregulated under inflammatory conditions.Overexpression of circ PRIM2 promoted the secretion of β-casein from mammary epithelial cells,and inhibited the proliferation and vitality of mammary epithelial cells,and regulate the activation of the NF-κB signaling pathway to suppresses the inflammatory response of mammary epithelial cells and promotes the expression of the circ PRIM2 parental gene in inflammatory breast epithelial cells induced by LPS and LTA.