Molecular Mechanism Of LncRNA And M6A Modification Regulation Mammary Epithelial Cell Injury In Bovine

Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus)are two common pathogenic microorganisms that cause clinical and subclinical bovine mastitis.In recent studies,long non-coding RNA(LncRNA)has been found to play an important role in the immune response that induced by microbial.However,the function of LncRNA in bovine mastitis remains unclear.The purpose of this study was to investigate the effects of injuries to bovine mammary epithelial cells induced by E.coli and S.aureus,analyze the role of LncRNA in cell injury and predict the possible molecular regulatory mechanism of cell injury.In addition,this study conducted genetic screening based on the sequencing data of LncRNA-seq and MeRIP-seq.The screened genes were verified by RT-qPCR,and it was found that the expression of LOC104974191 with hypomethylation increased in MAC-T cells stimulated by E.coli.Therefore,it is speculated that ALKBH5 is the major enzyme regulating the differential methylation of LOC104974191.In this study,RT-qPCR,FISH,MeRIP-qPCR,siRNA interference technology,construction of overexpression vector,Western Blot,flow cytometry and other experimental methods were used effectively.Specific results are as follows:1.Establishment bovine mammary epithelial cell inflammatory model in vitroBovine mammary epithelial cells were stimulated by S.aureus and E.coli that are inactivated by high temperature with MOI of 10:1.After 24h of stimulation,the expression levels of IL-1β,IL-6 and TNF-α were significantly increased and dectected by RT-qPCR(p<0.05),establishing the mastitis model in vitro succeesfully.And CCK8 was used to detect the growth activity of cells,and flow cytometry was used to detect the ROS level,cell cycle and apoptosis rate of cells.It was found that after stimulated by inactivated S.aureus and E.coli for 24h,cell-cycle arrest was observed,ROS and apoptosis rate was significantly increased.2.Analysis LncRNA-seq expression profile in bovine mammary epithelial cellsProfiling the LncRNA-seq data that was obtained in mastitis model in vitro,2342 differentially expressed LncRNAs were found in the E.coli stimulation group and 2313 differentially expressed LncRNAs were found in the S.aureus stimulation group.A total of 2225 LncRNAs were differentially expressed in both two stimulated groups.There were 268 significantly different LncRNAs in the E.coli and 238 significantly different LncRNAs in the S.aureus group.The bioinformatics analysis showed similar functions through predicting the signaling pathways targeted by the maternal genomes of E.coli and S.aureus.LncRNAs with significantly different expression levels were mainly enriched in PI3K-Akt signaling pathway,ErbB signaling pathway and tightly connected cell signaling pathway.Also,LncRNA-miRNA-mRNA interaction networks were successfully constructed.3.Detection of global methylation in bovine mammary epithelial cellsM6A methylation level of whole mRNA was detected in mastitis model in vitro,it was found that the m6A methylation level of whole cell was significantly increased in MAC-T cells stimulated by two inactivated bacteria for 24h(p<0.05).RT-qPCR was also used to detect the methylating modifying enzymes in the two stimulation groups,and it was found that experission of the methylase such as METTL3,METTL14 and WTAP were significantly increased(p<0.05),which was consistent with the trend of the increased global methylation level.The mRNA expression level of ALKBH5 was increased in the two inactivated bacteria stimulated groups(p<0.05),but there was no significantly difference of the expression of FTO in the two inactivated bacteria stimulation groups(p>0.05).4.Analysis LncRNA MeRIP-seq modification in bovine mammary epithelial cellsProfiling the MeRIP-seq of LncRNA data that was obtained in mastitis model in vitro,there were 298 m6A peaks within 112 LncRNA in the S.areus group and 920 different m6A peaks within 288 LncRNAs in the E.coli group.105 identical m6A peaks in both S.aureus and E.coli groups were hypermethylation,while 90 identical LncRNA m6A peaks were hypomethylation.The functional prediction of differentially m6A modified lncRNAs showed that they were mainly enriched in NF-κB signaling pathway,Wnt signaling pathway and mTOR signaling pathway.Conjoint analysis of MeRIP-Seq LncRNA-Seq showed that there were 139 LncRNAs in the S.aureus group and 20 LncRNAs in the E.coli group,which was different.It is speculated that these mRNA expression may be regulated by m6A methylation.5.Screening and identification of LOC104974191According to the sequencing data of LncRNA-seq and MeRIP-seq,differentially methylated LncRNA in the stimulation group were screened,and m6A modification on LOC104974191 was found to be significantly lower in the E.coli group.IGV software was used to visualize the methylation peak of LOC104974191.MeRIP-qPCR was used to detect that the m6A modification level of LOC 104974191 was decreased in the E.coli group,which was consistent with the sequencing results(p<0.05).6.ALKBH5 mediates methylation of LOC104974191 to regulate apoptosis of bovine mammary epithelial cells(1)Overexpression of ALKBH5 led to down-regulation of LOC104974191(p<0.05),while silencing ALKBH5 led to up-regulation of LOC104974191 with hypermethylation(p<0.05).It was proved that ALKBH5 affected m6A methylation level and expression level of LOC104974191.(2)After silencing LOC 104974191,the expression level of Cleaved-Caspase3 increased,while the expression level of PARP protein decreased(p<0.05).Moreover,silencing LncRNA promoted the overexpression of reactive oxygen species,inhibit cell activity,and improve the apoptosis rate of cells.It is proved that LOC 104974191 can promote ROS and increase cell apoptosis rate.(3)After ALKBH5 silencing,the expression levels of Caspase3 and Cleaved-Casepase3 were detected to increase,while the expression level of PARP protein was decreased(p<0.05).Moreover,ALKBH5 silencing could promote the reactive oxygen species,inhibit cell activity,and improve the apoptosis rate of cells.It was proved that ALKBH5 regulates the m6A modification level of LOC104974191,thereby promoting ROS and ultimately increasing apoptosis rate.In summary,we found that inactivated S.aureus/E.coli stimulated MAC-T cells to cause cell-cycle arrest,ROS and apoptosis rate was significantly increased.It was found that the m6A methylation level of whole cell was significantly increased and experission of the methylase such as METTL3,METTL14,WTAP and demethylase such as ALKBH5 were significantly increased.Profiling the data of LncRNA-seq and MeRIP-seq of LncRNA,it was found that total of 2225 LncRNAs were differentially expressed and 375 LncRNA were differentially m6A methylation,and it is predicted that these LncRNA can play important rolein regulating biological processes such as signal transduction and energy generation.This study also confirmed that ALKBH5 could regulate the expression of LOC104974191 and the methyl ation level of m6A,thus promoting ROS overexpression and ultimately promoting the increase of cell apoptosis rate and the decrease of cell growth activity.This study laid a foundation for the subsequent study of LncRNA-related mechanisms in the mammary gland of dairy cows.