Bovine mammary epithelial cells (bMECs) are part of the first line of defense against pathogens. In recent studies, bta-miR-223 has been reported to activate congenital and innate immunity against inflammatory damage during the pathogenesis of mastitis in dairy cows. The purpose of this study was to identify the regulatory mechanism of bta-miR-223 and its downstream target genes in inflammatory bMECs. A double luciferase reporter gene assay demonstrated that ras homolog family member B (RHOB) was the target gene of bta-miR-223. To further elucidate the role of bta-miR-223 in congenital immune responses, bta-miR-223 mimics (mimic/inhibitor) were transfected into bMECs stimulated with lipopolysaccharide (LPS), which activates the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect secreted inflammatory factors. Results showed that bta-miR-223 expression during inflammation in bMECs reduced the secretion of inflammatory factors by targeting RHOB and deactivation of NF-κB gene activity. Silencing RHOB inhibited LPS-induced inflammatory response in bMECs. Overall, bta-miR-223 attenuated LPS-induced inflammatory response, and acted as a negative feedback regulator via targeting RHOB, providing a novel avenue for mastitis treatment.

Bovine mastitis is an inflammatory disease caused by pathogenic microbial infection, trauma, or other factors. Its morbidity is high, and it is difficult to cure, causing great harm to the health of cows and the safety of dairy products. Susceptibility or resistance to mastitis in individual cows is mainly determined by genetic factors, including coding genes and non-coding genes. Long non-coding RNAs (lncRNAs) are a class of endogenous non-coding RNA molecules with a length of more than 200 nucleotides (nt) that have recently been discovered. They can regulate the immune response of humans and animals on three levels (transcription, epigenetic modification, and post-transcription), and are widely involved in the pathological process of inflammatory diseases. Over the past few years, extensive findings revealed basic roles of lncRNAs in inflammation, especially bovine mastitis. This paper reviews the expression pattern and mechanism of long non-coding RNA (lncRNA) in inflammatory diseases, emphasizes on the latest research progress of the lncRNA expression pattern and molecular regulatory mechanism in bovine mastitis, analyzes the molecular regulatory network of differentially expressed lncRNAs, and looks forward to the research and application prospect of lncRNA in bovine mastitis, laying a foundation for molecular breeding and the biological therapy of bovine mastitis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate post-transcriptional gene expression and several biological processes. Bovine mammary epithelial cells (bMECs) mediate critical immune responses in the mammary gland and the occurrence of mastitis. Current research focuses on miRNA regulation of bMECs, but the miR-375 regulatory mechanism in bMECs is unclear. This study explored the role of miR-375 by profiling the transcriptome of miR-375-silenced bMECs using RNA-seq and identifying differentially expressed mRNAs (DIE-mRNAs). There were 63 DIE-mRNAs, including 48 down-regulated and 15 up-regulated mRNAs between miR-375-silenced bMECs and the controls. The Kyoto encyclopedia of genes and genomes (KEGG) and Gene Ontology (GO) functional analysis showed that the DIE-mRNAs enriched nuclear receptor subfamily 4 group A member 1 (NR4A1) and protein tyrosine phosphatase non-receptor type 5 (PTPN5) anti-inflammatory genes of the mitogen-activated protein kinase (MAPK) signaling pathway. However, they showed an opposite trend to the expression of miR-375 silencing, suggesting that miR-375 promotes bMEC inflammation through the MAPK signaling pathway. The findings of this study provide a new reference for understanding the regulation of bMEC inflammation and cow mastitis.