| Staphylococcus aureus(S.aureus)bovine mastitis is an inflammatory disease caused by S.aureus infection in mammary gland tissue.Chronic antibiotic treatment not only increases antibiotic resistance of pathogenic bacteria,also increases the risk of over-consumption and abuse of antibiotics in human.Breeding new strains of S.aureus mastitis resistant cows is a promising solution for this problem.Micro RNAs(mi RNAs)plays a key regulatory role by binding to target genes to inhibit their translation or lead to their degradation.Therefore,this study aim to screen S.aureus mastitis resistance-related mi RNAs and explore the regulation mechanism of its target gene.In this study,somatic cell count(SCC),pathogen identification,milk sample PCR and paraffin section observation of mammary gland tissue were used to screen and identify healthy cows and S.aureus mastitis cows.RNA sequencing technology was used to construct mi RNAs differential expression profiles.Bioinformatics analysis technology was used to predict target genes of differentially expressed mi RNAs and annotate their functions.Then,S.aureus mastitis resistance-related mi RNAs were identified by RT-q PCR.Candidate target genes were obtained by bioinformatics prediction,and the target gene of S.aureus mastitis resistance-related mi RNAs was identified by mi RNA transfection,RT-q PCR,Western Blot,site-directed mutation in the seed region and dual luciferase reporter gene detection.At the same time,the inflammatory response of Mac-T cells was induced by S.aureus derived lipoteichoic acid(LTA)to construct the S.aureus mastitis cells model,and the effects of S.aureus mastitis resistance-related mi RNAs on the occurrence of the inflammatory response was investigated by mi RNA transfection and RT-q PCR.Cell culture,DNA transfection,RT-q PCR,and Western Blot were used to construct target gene interference and overexpression cell models,so as to analyze the expression changes of key proteins in the inflammatory response related signaling pathways.Using S.aureus to infect postpartum female BALB/c mice mammary gland,and then the RT-q PCR and paraffin section observation of mammary gland tissue and Western Blot were used to analysis of inflammatory cytokine m RNA expression level and expression changes of key proteins involved in the inflammatory response.The results showed that cow no.1,no.4 and no.8 were identified as healthy cows and cow no.2,no.6 and no.7 were identified as S.aureus mastitis cows.Compared with healthy cows,29 differentially expressed mi RNAs were significantly up-regulated and 19 differentially expressed mi RNAs were significantly down-regulated in the mammary gland tissues of S.aureus mastitis cows.Functional annotation analysis of candidate target genes of differentially expressed mi RNAs indicated that bta-mi R-223,bta-mi R-205,bta-mi R-21-5p,etc.were involved in signal pathways related to inflammatory response,and could be used as candidate marker-assisted selections of key factors of S.aureus mastitis resistance regulation.The transfection of 30 pmol/m L bta-mi R-223 mimic and bta-mi R-223 inhibitor into Mac-T cells for 48 h can successfully construct bta-mi R-223 overexpression and interference cell models,respectively.It was determined that 40 ?g/m L LTA induce Mac-T cells for 24 h could successfully construct inflammation cell model.Bta-mi R-223 overexpression and interference model and LTA-induced inflammation model were used in combination,it was found that the high expression of bta-mi R-223 in Mac-T cells could enhance their resistance to the inflammatory response induced by S.aureus-derived LTA.Compared with the control group,in bta-mi R-223 overexpressed cells,CBLB m RNA and protein expression levels were significantly down-regulated.When bta-mi R-223 was down-regulated,the expression of CBLB showed the opposite trend.After CBLB-3'-UTR original luciferase vector was co-transfected with bta-mi R-223 mimic,the relative dual luciferase activity dropped sharply.However,after co-transfection of CBLB-3'-UTR mutant luciferase vector with bta-mi R-223 mimic,there was no significant change in relative dual luciferase activity,proving that CBLB is a direct regulatory target of bta-mi R-223.After transfecting 4 ?g/m L CBLB overexpression and interference vector into Mac-T cells for 72 h,CBLB overexpression and interference cell models could be successfully constructed.In CBLB overexpression and LTA-induced Mac-T cells,the protein expression levels of PI3 K,AKT and the phosphorylated NF-?B p65 were significantly increased.In both CBLB interference and LTA-induced Mac-T cells,the expression levels of PI3 K,AKT and the phosphorylated NF-?B p65 protein were significantly decreased.These results suggest that bta-mi R-223 directly targets CBLB to participate in the regulation of S.aureus mastitis through the PI3K/AKT/NF-?B pathway.The dose of 108CFU/100 ?L S.aureus infecting the mammary gland of BALB/c mice for 48 h could construct the model of S.aureus mastitis in mice.Compared with the PBS treat group,the protein expression levels of CBLB,PI3 K,AKT and phosphorylation levels of NF-?B p65 protein were significantly increased in S.aureus mastitis mice.In summary,mi RNAs expression profiles were successfully constructed in the mammary gland tissues of healthy cows and S.aureus mastitis cows in this study and a total of 48 differentially expressed mi RNAs were identified in the two libraries.Functional annotation analysis of candidate target genes of differentially expressed mi RNAs indicated that bta-mi R-223,bta-mi R-205 and bta-mi R-21-5p,etc.could be used as candidate marker-assisted selections for S.aureus mastitis.Study on the potential resistance regulation mechanism of bta-mi R-223 has shown that bta-mi R-223 can down-regulate the expression levels of PI3 K,AKT and the phosphorylated NF-?B p65 protein by directly targeting CBLB,thus alleviating the LTA-induced inflammatory response in Mac-T cells. |
PDF Full Text Request