N6-Methyladenosine-Modified circRNA in the Bovine Mammary Epithelial Cells Injured by Staphylococcus aureus and Escherichia coli

Abstract

Mastitis is a common disease that hinders the development of dairy industry and animal husbandry. It leads to the abuse of antibiotics and the emergence of super drug-resistant bacteria, and poses a great threat to human food health and safety. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis in dairy cows and usually cause subclinical or clinical mastitis. CircRNAs and N6-methyladenosine (m6A) play important roles in immunological diseases. However, the mechanisms by which m6A modifies circRNA in bovine mammary epithelial cells remain poorly understood. The aim of our study was to investigate m6A-modified circRNAs in bovine mammary epithelial cells (MAC-T cells) injured by S. aureus and E. coli. The profile of m6A-modified circRNA showed a total of 1,599 m6A peaks within 1,035 circRNAs in the control group, 35 peaks within 32 circRNAs in the S. aureus group, and 1,016 peaks within 728 circRNAs in the E. coli group. Compared with the control group, 67 peaks within 63 circRNAs were significantly different in the S. aureus group, and 192 peaks within 137 circRNAs were significantly different in the E. coli group. Furthermore, we found the source genes of these differentially m6A-modified circRNAs in the S. aureus and E. coli groups with similar functions according to GO and KEGG analyses, which were mainly associated with cell injury, such as inflammation, apoptosis, and autophagy. CircRNA–miRNA–mRNA interaction networks predicted the potential circRNA regulation mechanism in S. aureus- and E. coli-induced cell injury. We found that the mRNAs in the networks, such as BCL2, MIF, and TNFAIP8L2, greatly participated in the MAPK, WNT, and inflammation pathways. This is the first report on m6A-modified circRNA regulation of cells under S. aureus and E. coli treatment, and sheds new light on potential mechanisms and targets from the perspective of epigenetic modification in mastitis and other inflammatory diseases.

Exosomes in Mastitis—Research Status, Opportunities, and Challenges

“…Seventh, the sorting mechanism of mastitis-related exosome cargo and the molecular mechanism mediating intercellular communication remain unclear; these areas of inquiry in mastitis research are still in their infancy. Three atlas-based articles describe the m6A modification of mRNA and circular RNA (circRNA) in MAC-T cells after E. coli or S. aureus infection [ 156 , 157 , 158 ]. Considering the function of m6A modification in promoting miRNA maturation [ 159 ], study into the role played by m6A in mastitis exosomal cargo sorting is warranted.…”

The Role of N6-Methyladenosine in Inflammatory Diseases

“…In Salmonella typhimurium infection, YTHDF2 depletion promoted H3K27me3 demethylation of multiple inflammatory cytokines in the MAPK and NF- κ B signaling pathways, such as IL-6 and IL-12B, and subsequently enhanced its transcription [ 158 ]. Moreover, cell injury induced by E. coli and S. aureus has also been proved to be associated with m 6 A modification, and differentially expressed genes are mainly enriched in inflammation, apoptosis, and autophagy [ 47 , 159 ].…”

Emerging roles of noncoding micro RNAs and circular RNAs in bovine mastitis: Regulation, breeding, diagnosis, and therapy

“…Additionally, circRNAs are found to be highly expressed in specific tissues and stages throughout animal genome, offering a potential biomarker or treatment target for disease detection ( Wu et al, 2022 ). Moreover, considering its unique features, modified investigation methods have been proposed for further bioinformatical analysis and more functions have been predicted and verified during the interaction between pathogen and host, thus improving the insight of animal breeders ( Wang J. P. et al, 2022 ; Xu et al, 2022 ). Research on animal circRNAs has become a hotspot in genetics and molecular biology with advances in techniques ( Jeck and Sharpless, 2014 ).…”

“…Among the three regulatory mechanisms mentioned above, miRNA sponging is the most frequently demonstrated action found in the inflammation of bovine mammary gland, which we will discuss in detail later. In addition, epigenetic modifications (N 1 -methyladenosine, 2′-O-ribosemethylation, inosine, 5-methylcytidine, N 6 -methyladenosine (m 6 A) and pseudouridine) in RNA molecules have captured researcher’s attention in recent years, and the functions and applications of these unique molecules have also been expanded upon ( Wu et al, 2021 ; Xu et al, 2022 ). Following the deepening of circRNA research, the regulatory mechanism of ncRNA network in animal biological process will be clearer.…”

“…However, there are very few studies on epigenetic modifications in mastitis-related circRNAs. In a recent study conducted by Xu et al (2022) , RNA immunoprecipitation and high-throughput sequencing (MeRIP-seq) were used for the first time to profile circRNAs regulated by m6A modification in mammary epithelial cells and the different pathological patterns between S. aureus and E. coli treatment were compared. Notably, the m 6 A-modified circRNAs, differentially expressed in the two groups were found to be highly similar in functional predictions, including immunity, cell junctions, growth metabolism, and resistance to bacterial invasion, indicating the compatibility and biomarker potency of circRNAs in mastitis.…”