| Mastitis in dairy cows is a complex disease caused by infection of mammary tissue by pathogenic microorganisms,which has caused great economic losses to modern dairy industry.miRNA has been shown to regulate the expression of genes related to mastitis in dairy cows,which has a very important role in the regulation of intrinsic immune response and inflammatory immunity.In this study,we intend to further investigate the candidate miR-375 at the molecular and cellular levels,reveal the expression pattern of miR-375 in dairy mammary gland tissue and dairy mammary epithelial cells(bMEC)during the inflammatory response,identify the expression of miR-375 on key genes of the intrinsic immune signaling pathway and its effect on the inflammatory response process of bMEC,and initially investigate the role of miR-375 in LPS-induced inflammatory response in bMEC.The main findings are as follows:(1)Analysis of miR-375 expression pattern in mammary tissue and bMEC inflammatory response in mastitis cowsIn this study,mammary tissues from healthy and mastitis cows were collected,while LPS was successfully used to induce an inflammatory response in bMEC.The expression of IL-6,IL-8,TNF-αand miR-375 in tissues and cells were detected,and the results showed that the expression of inflammatory factors IL-6,IL-8 and TNF-α were significantly up-regulated in mammary tissues and cells at different times of inflammation induction in mastitis cows,and the expression level of miR-375 was significantly down-regulated in both tissues and cells,which laid the foundation for further studies to verify their molecular mechanisms.(2)Identification of miR-3 75 target genes affecting the intrinsic immune inflammatory response in dairy cows with mastitisUsing bioinformatics analysis,the possible candidate target genes of miR-375 were predicted,and a total of 13 genes,including TAB1,MAP3K7,SCOS5,TRADD,CASP8,NLK,IKBKE,TRAF6,YAP1,SP1,YBX1,JAK2 and KLF5,were initially screened as possible target genes of miR-375.In this study,we constructed 3"UTR recombinant vectors of the above 13 potential target genes and identified the regulatory relationship between miR-375 and the above predicted target genes by dual luciferase reporter gene assay,and the results showed that there was a decrease in enzyme activity in the experimental group of YAP1,KLF5 and YBX1,and there was no significant change in the enzyme activity of other candidate target genes.And the efficiency of miR-375 to inhibit the enzyme activity of YBX1 target gene identified reached more than 30%,and YBX1 was tentatively considered as the target gene of miR-375.(3)Effect of miR-375 on the inflammatory response of bMECTo investigate the effects of elevated and decreased miR-375 expression on the mRNA expression of related pathway genes and inflammatory factor genes,mimics and inhibitors of miR-375 were transfected into bMEC,and the mRNA expressions of target gene YBX1,downstream pathway genes and inflammatory factor genes were examined after LPS induced inflammatory response in bMEC,respectively.The results showed that in bMEC,miR-375 negatively regulated the expression of the target gene YBX1,which was consistent with the identification of the target gene of miR-375.During the LPS-induced inflammatory response in bMEC,overexpression of miR-375 could target and inhibit the expression of YBX1 gene and significantly elevate the expression of the pro-apoptotic factor BAD and the inflammatory factors IL-6 and IL-8,and these results suggest that miR-375 may promote the apoptosis of bMEC during the inflammatory response. |
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