Bta-miR-223 Targeting CBLB Contributes to Resistance to Staphylococcus aureus Mastitis Through the PI3K/AKT/NF-κB Pathway
Shuo Han, Xinli Li, Juan Liu, Ziwen Zou, Lin Luo, Rui Wu, Zhihui Zhao, Changyuan Wang, Binglei Shen
Abstract
Staphylococcus aureus (S. aureus)bovine mastitis is a kind of inflammatory disease caused by S. aureus infection in mammary gland tissues. MicroRNA (miRNA) plays a regulatory role by binding to target genes to inhibit their translation or lead to their degradation. Therefore, this study aim to identify S. aureus mastitis resistance-related miRNAs and explore regulatory mechanism of its target gene. Methods such as somatic cell count, pathogen identification, milk sample PCR, paraffin section observation, high-throughput sequencing, bioinformatics, miRNA transfection, DNA transfection, RT-qPCR, Western Blot, dual luciferase reporter gene detection were used in this study. The results showed that 48 differentially expressed miRNAs(DE-miRNAs) were identified in the breast tissues between heathy and S. aureus mastitis cows. Bta-miR-223, bta-miR-205, bta-miR-21-5p, etc. could be used as candidate marker-assisted selections of resistance regulation of S. aureus mastitis. The overexpression of bta-miR-223 enhanced the resistance of Mac-T cells to the inflammation induced by S. aureus-derived lipoteichoic acid(LTA). CBLB was identified as a direct target gene of bta-miR-223. In CBLB-overexpressed and LTA-induced Mac-T cells, the protein expression levels of PI3K, AKT and phosphorylated NF-κB p65 were significantly increased. In CBLB-interfered and LTA-induced Mac-T cells, they were significantly decreased. Compared with the PBS group, after infecting 108CFU/100μL S. aureus for 48 h, the protein expression levels of CBLB, PI3K, AKT and phosphorylated NF-κB p65 in the breast tissues of mice were significantly increased. In summary, bta-miR-223, bta-miR-205, bta-miR-21-5p, etc. could be used as candidate marker-assisted selections of key factors in the resistance regulation of S. aureus mastitis. Bta-miR-223 participate in the resistance regulation of S. aureus mastitis through the PI3K/AKT/NF-κB pathway by directly targeting CBLB.