Regulatory Mechanism Of RNA M6A Modification On Inflammatory Response Of Bovine Mammary Epithelial Cells Induced By Staphylococcus Aureus/Escherichia Coli

Mastitis is the most common disease in dairy cows,with high incidence and difficult treatment,which have caused significant losses in the dairy industry.Staphylococcus aureus and Escherichia coli are the two most common pathogenic microorganisms that induce mastitis in dairy cows.Bovine mammary epithelial cells play an important role in the defense against S.aureus and E.coli infections.Throgh the regulation of signal pathways such as toll-like receptors,MAPK,and apoptosis,it secretes varieties cytokines such as inflammatory factors and chemokines,exerting an immune response.N6-methyladenosine methylation is a kind of RNA modification that exists widely in bacteria and eukaryotes.Through writers,erasers and readers,m6 A methylation-related enzymes dynamically regulate this process,it plays an important role in the occurrence and development of various diseases.However,the role and mechanism of methylation of m6 A in mastitis is still unclear.In this study,we established the inflammation model of bovine mammary epithelial cells induced by S.aureus/E.coli,drew the methylation profile of m6 A,and identified the differentially methylated TLR4 molecule.Silenced and overexpressed ALKBH5,the changes of TLR4 m6 A mediated by ALKBH5 were analyzed to clarify the molecular mechanism of ALKBH5 mediated TLR4 m6 A regulating the inflammatory response of mammary epithelial cells,which laid a foundation for the study of the role of m6 A methylation in the prevention and control of dairy cow mastitis.The main results are as follows:1.Establishment of inflammation model of bovine mammary epithelial cellsThe MAC-T cells were induced by S.aureus/E.coli with infection ratio of 10:1.After24 hours,the cells and supernatant were collected respectively.The expression of inflammatory factors IL-1β,IL-6 and TNF-α was detected by RT-qPCR and ELISA.The expression of inflammatory factors was significantly increased(p < 0.05),indicating that the in vitro infection model was established successfully.2.Differential expression of methylating modifying enzymes in bovine mammary epithelial cellsBased the above model,the mRNA expressions of m6 A modified methyltransferase METTL3,METTL14,WTAP and demethylase ALKBH5,FTO were detected respectively.It was found that the mRNA expression levels of m6A modified methyltransferase METTL3,METTL14,WTAP and demethylase ALKBH5 in MAC-T cells were increased(p<0.05),while the mRNA expression levels of FTO in MAC-T cells were not significantly different(p>0.05).3.Profiling of the m6 A modification in bovine mammary epithelial cellsMeRIP-seq analysis was performed on bovine mammary epithelial cells induced by inactivated S.aureus/E.coli.In S.aureus group,1006 significantly different m6 A methylation peaks were found in 844 mRNAs;in E.coli group,2904 significantly different m6 A methylation peaks were found in 2101 mRNAs.In addition,we also compared and analyzed the differences of the methylation peaks of m6 A between S.aureus group and E.coli group.In these two groups,the methylation peaks of 18 mRNAs were hypermethylation,and the methylation peaks of 390 mRNAs were hypomethylation.Bioinformatics analysis showed that the differentially methylated m6 A mRNA was mainly enriched in TGF-β signaling pathway,NF-κB signaling pathway,Hippo signaling pathway,MAPK signaling pathway and other important biological processes.Combined analysis of MeRIP-seq and mRNA-seq showed that there were 61 mRNAs in the S.aureus group and212 mRNAs in the E.coli group,and their expression may be related to the m6 A modification.4.Screening and identifing of TLR4According to the sequencing data of m6 A methylation modification profile,we screened the mRNA with different m6 A methylation peaks and found that TLR4 was the mRNA with significantly low m6 A modification in S.aureus group and E.coli group.IGV software was used to visualize the methylation sites of TLR4 m6 A in the sequencing data.MeRIP-qPCR confirmed that the methylation level of TLR4 in S.aureus group and E.coli group was significantly reduced(p < 0.05).5.ALKBH5 mediates the methylation of TLR4 and regulates the inflammatory response of bovine mammary epithelial cellsBased on the MeRIP-seq data,this study found that the m6 A modification of the molecule TLR4 was down-regulated in S.aureus/E.coli-infected MAC-T cells(p <0.05).At the same time,it was based on the previous level of demethylase mRNA.It was verified that ALKBH5 is speculated to be a demethylase that changes TLR4 to m6A methylation modification.This study used MeRIP-qPCR,si RNA interference technology,the construction of overexpression vector and RT-qPCR,Western Blot and other experimental methods to verify.The findings are as follows:(1)The results showed that overexpression of ALKBH5 can lead to down-regulation of TLR4 m6 A levels,mRNA levels,and protein levels(p <0.05),inhibitor of ALKBH5 can lead to up-regulation of TLR4 m6 A levels,mRNA levels,and protein levels(p <0.05),which is proved that ALKBH5 can affect the m6 A,mRNA and protein changes of TLR4;(2)The results showed that overexpression of ALKBH5 can lead to the mRNA and the phosphorylated protein level of JNK,ERK,p38 and p65 in MAPK/NF-κB signaling pathways down-regulated(p < 0.05);inhibitor of ALKBH5 can lead to the mRNA and the phosphorylated protein level of JNK,ERK,p38 and p65 in MAPK/NF-κB signaling pathways up-regulated(p < 0.05),which was proved that ALKBH5 can mediate MAPK/NF-κB signaling pathway through TLR4;(3)The results showed that overexpression of ALKBH5 can lead to the mRNA levels and protein level of inflammatory factors IL-1β,IL-6,TNF-α down-regulation(p <0.05);inhibitor of ALKBH5 can lead to the mRNA levels and protein level of inflammatory factors IL-1β,IL-6,TNF-α up-regulation(p <0.05);which was proved that ALKBH5 regulates the release of inflammatory factors by mediating TLR4/MAPK/NF-κB.In summary,this study found the difference in the expression of m6 A methylationrelated enzymes in MAC-T cells infected with S.aureus/E.coli,while found 1006 different m6 A methylation peaks in S.aureus group,2904 different m6 A methylation peaks in E.coli group.This study also confirmed that ALKBH5 affects the expression of m6 A methylation,mRNA and protein level of TLR4,which in turn affects the MAPK/NF-κB signaling pathway,then affects the expression and release of inflammatory factors.The research laid a foundation for the follow-up study on the mechanism of mastitis about m6 A.