| Mastitis is one of the most common diseases in dairy cow,which seriously affects the economic benefits of dairy farms.In order to breed dairy cow with strong mastitis resistance,it is particularly important to screen the mastitis-related gene and clarify its molecular regulation mechanisms.The gene expression profiles of mastitis cows is greatly affected by many factors,such as the types of pathogen,the dose of pathogen infection,and the time of infection.Up to date,the molecular mechanism of mastitis in dairy cows has not been fully understood.Therefore,in order to further explore the key gene and clarify the molecular regulation mechanism of cow mastitis,in this study,the experimental mastitis models of E.colitype and S.aureustype were successfully constructed by long-term?7d?infection of the udders of Holstein cows with E.coli and S.aureus?5mL 1×105 CFU/mL?,respectively.On this basis,the RNASeq technique was used to perform transcriptome and miRNA sequencing of mammary tissues and peripheral blood at different times of pathogen induction,then,we screened the differentially expressed mRNA or miRNA,and perform the combined analysis of mRNA-miRNA in mammary gland.In addition,many molecular and cell biology research methods,including miRNA overexpression/silencing,qPCR,Western Blotting,ELISA,flow cytometry ect.,were used to investigate the molecular mechanism of bta-miR-146a in LPS-induced mammary epithelial cells?bMEC?.The main results are as follows:?1?The protein expression levels of TLR4,IRAK1,TRAF6,NF-?B and TNF?in the mammary tissues with E.coli-type or S.aureus-type mastitis were significantly up-regulated compared with the control group,indicating that both types of mastitis models initiate TLR4/NF-?B-mediated innate immune responses and inflammatory responses.?2?Gene expression profiles and differentially expressed genes in mammary tissues infected by E.coli or S.aureus were obtained.Compared with the control group,the results of transcriptome sequencing showed that there were 2388 differentially expressed mRNAs?DIE-mRNA?and 305 differential expression miRNA?DIE-miRNA?in the E.coli-induced mammary tissues.Meanwhile,there were 2642 DIE-mRNAs and 279 DIE-miRNAs in S.aureus-induced mammary gland,in which,LOC785756,AQP5,bta-miR-202 and bta-miR-2357 may be useful molecular markers of E.coli-type mastitis,in which,many genes,including DDC,C1QTNF3,CCDC60,TCTEX1D1,ZNF358,FNDC1,COL11A1 and bta-miR-2335 may be molecular markers of S.aureus-type mastitis.?3?The mRNA and miRNA expression profiles of the mammary tissues with E.coli type and S.aureus type mastitis are significant different.Compared with the S.aureus induced group,total of 627 DIE-mRNA and 21 DIE-miRNAs were down-regulated,482 DIE-mRNA and 70 DIE-miRNA were up-regulated in mammary tissues of E.coli induced group,suggesting that these DIE-mRNA and DIE-miRNA may play different roles in the two types of mammary gland inflammatory response,and may also be related to the degree of inflammation and the immune response process.?4?The miRNA-mRNA regulatory network of mammary tissue in immune regulation and apoptosis was obtained through the combined analysis of miRNA and transcriptome.In terms of animal immunity,miRNAs mainly regulate the mRNA related to TLRs signaling pathways,CD family,cytokine or cytokine receptor.In terms of apoptosis,four mRNAs?BAX,BCL2A1,BCL2L14,PYCARD?and five miRNAs?bta-miR-1486,bta-miR-885,bta-miR-30e-5p,bta-miR-139 and bta-miR-1291?are present in regulatory networks of E.coli types or S.aureus types of mastitis.?5?The gene expression profiles and differentially expressed genes of peripheral blood of different times of infection in E.coli type or S.aureus type mastitis cows were obtained.Among them,6 known genes?ZBTB39,AMER1,KLHL9,ABHD13,FZD5,TET2?and 3new genes?CownewGene1702,CownewGene4598 and CownewGene4025?might be used as blood molecular markers of dairy cow with E.coli-type mastitis.Meanwhile,the HBM,HBG,DEFB5,CCR3,bta-miR-1301 and bta-miR-2284r might be used as blood molecular markers of dairy cow with S.aureus-type mastitis.?6?Functional annotation of DIE-miRNA and DIE-mRNA of mammary tissue and peripheral blood of E.coli-and S.aureus-type mastitis were obtained.The DIE-mRNA and DIE-miRNA metioned above might be involved in many biological processes including genetic information processing,biological systems,diseases,metabolism,cellular processes and environmental information processing,and might participate in multiple immune system-related signaling pathways including Toll-like receptor signaling pathway,chemokine signaling pathway,cytokine and cytokine receptor signaling pathways,which regulate the process of cow mastitis.?7?TLR4 and TRAF6 are target genes of bta-miR-146a.Bta-miR-146a directly targets TLR4 and TRAF6 gene,and significantly inhibits the mRNA and protein expression of TRAF6 during inflammatory response in LPS-induced bMEC.In the case of the TLR4 gene,bta-miR-146a significantly inhibits the expression of the TLR4 gene in the early stage of the bMEC inflammatory response,while the inhibition efficiency in the late stage of the inflammatory reaction is not significantly different,indicating that other factors except for bta-miR-146 also regulates the expression of TLR4 gene.?8?LPS could activate TLR4/NF-?B signaling pathway in bMEC,which increases the expressions of TLR4,TRAF6 and the transcription factor NF-?B,consequently promotes the expression of bta-miR-146a.The higher expression of bta-miR-146a negative feedback inhibits the expression of target genes TLR4 and TRAF6,thereby inhibiting the expression of NF-?B and the secretion of inflammatory factors IL-6,IL-8 and TNF-?,and alleviating the inflammatory process of bMEC.?9?bta-miR-146a promotes apoptosis of bMEC in the inflammatory response.The above results should lay a foundation for elucidating molecular regulation network of mastitis in dairy cows,and also presents the molecular materials for molecular diagnosis,biological therapy and mastitis resistance molecular breeding,which has potential application value. |
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