| Bovine mastitis is one of the most prevalent diseases in dairy industry and has a very high clinical incidence.After pathogenic bacterium in the environment invade the mammary gland of bovine to break through the physical barrier formed by the tight junction of the mammary epithelium,they rapidly expand in the mammary gland which can destroy the blood-milk barrier,thereby triggering the inflammatory reaction of the bovine mammary gland.Recent studies have reported that long non-coding RNAs(lncRNAs)play multiple regulatory role in a variety of inflammatory processes,but their involvement in the inflammatory response of bovine mammary gland have rarely been reported.In the early stage of the laboratory,a high-throughput sequencing revealed a piece of lncRNA that was significantly down-regulated in a heat-inactivated E.coli-induced bovine mastitis cell model.This lncRNA was at the antisense strand of leucine-rich repeat-containing protein 75A(LRRC75A)and had partial base complementation with LRRC75 A,so named this lncRNA as LRRC75 A antisense lncRNA1(LRRC75A-AS1).In this study,the CRISPR/Cas9 system was used to knockout LRRC75A-AS1 in mammary gland epithelial cells.The roles and mechanism in mammary gland epithelial microscopy were studied by a series of molecular biologic methods and means.The experimental results were as follows:1.In the inflammatory state,the expression of LRRC75A-AS1 in the mammary gland and mammary epithelial cells of bovine was significantly higher than that of the normal control group.Knockout of LRRC75A-AS1 in mammary epithelial cells can induce morphological changes,promote the secretion of ?-casein in mammary epithelial cells,inhibit the proliferation of mammary epithelial cells,induce DNA damage,and enhance the expression of cell tight junction proteins Claudin-1,Occludin and ZO-1;knockout of LRRC75A-AS1 reduced the permeability of the mammary epithelial membrane and inhibited bacterial adhesion and invasion.In addition,knockout of LRRC75A-AS1 inhibited the expression of inflammatory cytokines and inhibited the activation of the NF-?B pathway.2.RNAplex software predicted that LRRC75A-AS1 binds to LRRC75 A in a base-complementary region.Knockout of LRRC75A-AS1 significantly inhibited the expression of LRRC75 A mRNA.In addition,interfering with LRRC75 A caused the decrease of membrane permeability of mammary epithelial cells,increased expression of tight junction proteins Claudin-1,Occludin and ZO-1,a significant decrease in bacterial adhesionand invasion,and inhibited NF-?B signaling pathway.When the expression of LRRC75 A in the mammary epithelium was overexpressed,the opposite result was obtained.The above results indicated that the LRRC75 A gene was involved in the regulation of mammary epithelial cell function by LRRC75A-AS1.In summary,LRRC75A-AS1,which is highly expressed in inflammatory state,can affect the basic biological characteristics and functions of mammary gland epithelial cell morphology,proliferation,and ?-casein secretion;furthermore,LRRC75A-AS1 regulated the expression of tight junction protein through LRRC75 A,affected cell permeability and bacterial invasion and adhesion,and participated in the inflammatory process of mammary epithelial cells by regulating the activation of NF-?B pathway.The results of this study provide new ideas for the study of the pathogenesis of dairy cow mastitis. |
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