Molecular Regulatory Function Of MiR-382 In Bovine Mastitis

There are many inducing factors for mastitis,which are generally caused by pathogenic microorganism infection.When pathogenic microorganisms invade the mammary gland of dairy cows,it will cause local or systemic inflammation,reduce the output and quality of milk,and cause huge economic losses to the dairy industry.Therefore,it is urgent to strengthen the research on the molecular regulation mechanism of bovine mastitis,and develop molecular breeding technology against bovine mastitis,so as to effectively improve the anti-mastitis ability of dairy cows.In recent years,scholars have done a great deal of research on the susceptibility and disease resistance of bovine mastitis,but its molecular regulation mechanism has not been fully clarified,and there are still many functional genes and their regulation functions that need to be further explored.Therefore,in this study,bovine mammary epithelial cells were induced by lipopolysaccharide(LPS),and the inflammatory model of bovine mastitis cells was successfully constructed.After over-expression or silence,the molecular regulation function of miR-382 in dairy mastitis was clarified by quantitative real-time PCR(qRT-PCR),dual luciferase reporter gene assay,enzyme linked immunosorbent assay(ELISA),Western Blot experiment,CCK-8,and Flow cytometry assay.The following main experimental results were obtained:(1)After over-expression of miR-382,134 up-regulated and 40 down-regulated mRNA were obtained through RNA-Seq technology;The results of functional enrichment show that miR-382 is mainly involved in regulating more than 30 signaling pathways such as T cell receptor signaling pathway,MAPK signaling pathway,Rapl signaling pathway,IL-17 signaling pathway and PI3K/AKT signaling pathway,affecting biological processes such as cell communication,signal transduction and response to stimuli,and has a significant impact on extracellular space,extracellular region and extracellular matrix,and peptidase regulatory activity;(2)At LPS induced bovine mammary epithelial cells 0,3,6,12 and 24 hours,the expression of miR-382 and pro-inflammatory cytokines IL-6,IL-1β,and IL-8 were detected by qRT-PCR technology.The results showed that compared with 0 h,the expression of miR-382 increased significantly after LPS induction 3 and 12 h(p<0.001),the expression of induction 6 h was the highest(p<0.0001),and the expression of 24 h was not significant adjusted(p>0.05);The expression of IL6,IL-1β,and IL-8 were significantly up-regulated after LPS-induced 3,6,12 and 24 h(p<0.01),indicating that this study has successfully constructed an inflammation model of bovine mammary epithelial cells;(3)The target genes of miR-382 were predicted by bioinformatics method.A total of 18 candidate target genes such as PTEN,NKAP,and GRK5 were screened,and the 3’UTR-psiCHECKTM-2 wild-type vectors of 18 candidate target genes were successfully constructed,and candidate target genes were identified by dual luciferase reporter gene assay,qRT-PCR and Western blot experiments.The results showed that miR-382 could directly target the 3’UTR region of GRK5 gene,inhibiting the mRNA and protein expression level of GRK5 gene;(4)The research results of the function of miR-382 in LPS-induced bovine mammary epithelial cells show that LPS can active the PI3K/AKT/NF-κB signaling pathway;Over-expression of miR-382 can inhibit the expression of PI3K,AKT and NF-κB of LPS-induced bovine mammary epithelial cells,thus inhibiting the secretion of pro-inflammatory cytokines IL-6,IL-1β,and IL-8,enhancing cell viability and inhibiting cell apoptosis;After inhibiting miR-382,the opposite results were obtained to over-expression of miR-382.To sum up,the expression of miR-382 in LPS-induced bovine mammary epithelial cells is up-regulated,which can target inhibit the expression of GRK5 gene,inhibit the inflammatory response and apoptosis of LPS-induced bovine mammary epithelial cells,and enhance cell viability.The research results reveal the function of miR-382 in bovine mastitis at the cellular level,which provides a reference basis for the analysis of molecular regulatory network of bovine mastitis and the breeding of anti-mastitis molecular.