Bta-miR-223 Targeting CBLB Contributes to Resistance to Staphylococcus aureus Mastitis Through the PI3K/AKT/NF-κB Pathway

Abstract

Bovine mastitis is an inflammatory condition of the mammary gland often caused by (Staphylococcus aureus) S. aureus infection. The aim of this study was to identify mastitis-related miRNAs and their downstream target genes, and therefore elucidate the regulatory mechanisms involved in disease progression and resistance. Three healthy and three mastitic cows were identified on the basis of the somatic cell count and bacterial culture of their milk, and the histological examination of udder tissues. High-throughput RNA sequencing and bioinformatic analyses revealed that 48 differentially expressed miRNAs (DEMs) in the mastitic udder tissues relative to the healthy tissues. Among 48 DEMs, the expression level of bta-miR-223 was the most up-regulated. Overexpression of the bta-miR-223 in Mac-T cells mitigated the inflammatory pathways induced by S. aureus-derived lipoteichoic acid (LTA). The Cbl proto-oncogene B (CBLB) was identified as the target gene of bta-miR-223, and the direct binding of the miRNA to the CBLB promoter was confirmed by dual luciferase reporter assay using wild-type and mutant 3'-UTR constructs. Furthermore, overexpression of CBLB in the LTA-stimulated Mac-T cells significantly upregulated PI3K, AKT, and phosphorylated NF-κB p65, whereas CBLB knockdown had the opposite effect. Consistent with the in vitro findings, the mammary glands of mice infected with 10⁸CFU/100 μL S. aureus showed high levels of CBLB, PI3K, AKT, and p-NF-κB p65 48 h after infection. Taken together, bta-miR-223 is a predominant miRNA involved in mastitis, and bta-miR-223 likely mitigates the inflammatory progression by targeting CBLB and inhibiting the downstream PI3K/AKT/NF-κB pathway.

Keywords: CBLB; Staphylococcus aureus; bovine mastitis; bta-miR-223; resistance regulation mechanism.

Figure 1

Isolation and culture of pathogenic bacteria of mastitic udders. (A) E. coli, (B) Streptococcus, and (C) S. aureus.

Figure 2

Histological examination of mammary gland tissues of healthy and mastitic cows.

Figure 3

Identification of mastitis-related miRNAs. (A) Significantly enriched KEGG pathways of predicted target genes. (B) Expression levels of mastitis-related DEMs.

Figure 4

Establishment of bta-miR-223 overexpressing and silenced cell lines. (A,B) bta-miR-223 mimic transfected Mac-T cells for 48 h, (C,D) bta-miR-223 inhibitor transfected Mac-T cells for 48 h, (E) effect of bta-miR-223 mimic on the expression of bta-miR-223, (F) effect of bta-miR-223 inhibitor on the expression of bta-miR-223. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

LTA-stimulated inflammation in Mac-T cells. (A) LTA stimulates Mac-T cells for 3 h, (B) LTA stimulates Mac-T cells for 6 h, (C) LTA stimulated Mac-T cells for 12 h, (D) LTA stimulated Mac-T cells for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Effect of bta-miR-223 mimic (A) and bta-miR-223 inhibitor (B) on LTA-stimulated inflammatory factor mRNA expression levels in Mac-T cells. ***p < 0.001 compared to PBS group, ^#p < 0.05 compared to LTA group, ^##p < 0.01 compared to LTA groups.

Figure 7

Screening of target genes of bta-miR-223. (A) The effect of bta-miR-223 mimic on the mRNA levels of its candidate target genes, (B) the effect of bta-miR-223 inhibitor on the mRNA levels of its candidate target genes, (C) the effect of bta-miR-223 overexpression on the expression level of CBLB protein. *p < 0.05; **p < 0.01.

Figure 8

Identification of the targeting relationship between bta-miR-223 and CBLB. (A,B) bta-miR-223 mimic transfected HEK-293T cells for 48 h, (C,D) bta-miR-223 inhibitor transfected HEK-293T cells for 48 h, (E) dual luciferase reporter gene test result.

Figure 9

Establishment of CBLB overexpressing and silenced cell lines. (A,B) CBLB silenced vector transfecting into Mac-T cells for 72 h, (C,D) CBLB overexpressing vector transfecting into Mac-T cells for 72 h, (E) the mRNA expression of CBLB after transfecting CBLB silenced vector for 72 h, (F) the mRNA expression of CBLB after transfecting CBLB overexpressing vector for 72 h. **p < 0.01.

Figure 10

Effect of CBLB overexpression on the PI3K/AKT/NF-κB p65 pathway in LTA-stimulated Mac-T cells. (A) Immunoblot showing expression levels of CBLB, PI3K, AKT, and NF-κB p65. Quantitative comparison of (B) CBLB, (C) PI3K, (D) AKT, (E) NF-κB p65, and (F) p-NF-κB p65. **p < 0.01.

Figure 11

Effect of CBLB silence on the PI3K/AKT/NF-κB p65 pathway in LTA-stimulated Mac-T cells. (A) Immunoblot showing expression levels of CBLB, PI3K, AKT, and NF-κB p65. Quantitative comparison of (B) CBLB, (C) PI3K, (D) AKT, (E) NF-κB p65, and (F) p-NF-κB p65. *p < 0.05; **p < 0.01.

Figure 12

Establishment of S. aureus-induced mastitis model in BALB/c mice. Representative images of mammary glands tissue sections of (A) PBS control and S. aureus-inoculated mice after (B) 6 h, (C) 12 h, (D) 24 h, and (E) 48 h after infection. Expression levels of pro-inflammatory cytokine mRNAs in the mammary glands after (F) 6 h, (G) 12 h, (H) 24 h, and (I) 48 h after infection. *p < 0.05; **p < 0.01.

Figure 13

The CBLB/PI3K/AKT/NF-κB p65 pathway is activated in the S. aureus-infected mammary gland tissues of BALB/c mice. (A) Immunoblot showing expression levels of CBLB, PI3K, AKT, and NF-κB p65. Quantitative comparison of (B) CBLB, (C) PI3K, (D) AKT, (E) NF-κB p65, and (F) p-NF-κB p65. *p < 0.05; **p < 0.01.