| Bacterial diarrhea in piglets is a common infectious disease in large-scale pig farms,among which E coli F18 is the main pathogen causing bacterial diarrhea of weaned piglets,the mechanism of pig resistance regulation remains to be further elucidated.With the continuous research in molecular biology research new functions and regulatory mechanisms of RNA have been discovered.N6-methyladenosine（m6A）is one of the most abundant modifications in RNA methylation,and studies have shown that m6A methylation modifications play an important role in biological processes such as viral replication,bacterial infection and immune response.However,there is a lacunae regarding the post-transcriptional regulatory mechanism of m6A modification on E.coli resistance in porcine F18.In this study,the porcine intestinal epithelial cell line（IPEC-J2）was used as the main research model.Firstly,we systematically verified the relationship of porcine m6A methylesterase WTAP expression on the resistance of E.coli F18.Secondly,comparative transcriptomic sequencing was used to perform a joint screening of differentially expressed genes between before and after WTAP interference group and before and after E.coli F18 infection group to identify the key target genes regulated by WTAP.Different experiments such as MeRIP-qPCR,Point mutation,Dual luciferase,RIP-qPCR were used to explore the regulatory mechanism of porcine WTAP-mediated m6A modification on the expression of target genes,which ultimately affects the susceptibility of IPEC-J2 cells to E.coli.The main results are as follows:1.In order to explore the relationship between WTAP expression and E.coli infection,we used qRT-PCR to detect the expression of WTAP in duodenal and jejunal tissues of resistant and susceptible types of E.coli F18 infection in Meishan pigs,and found that the expression of WTAP in the intestinal tissue of the resistant type was significantly higher than that of the susceptible type（P<0.01）;Both WTAP gene mRNA and protein expression were highly significantly upregulated after stimulation of IPEC-J2 cells using F18ab and F18ac strains（P<0.01）;IFA showed that WTAP protein was mainly localized in the nucleus,and its distribution level increased significantly after E.coli F18 infection.In this study,the IPEC-J2 cell line stably interfered by WTAP lentivirus was successfully constructed,and the interference efficiency was 53.68%.The effect of WTAP gene expression changes on the adhesion level of E.coli F18 in vitro were evaluated by PILIN quantification,colony counting,Gram staining,Indirect Immunofluorescence Assay and scanning electron microscopy.The results showed that after knockdown of WTAP gene in IPEC-J2 cells,the number of E.coli F18 adherent cells was significantly increased,indicating that WTAP may act as a key m6A methylation enzyme regulating the IPEC-J2 to E.coli F18,and the up-regulated expression of WTAP is beneficial to improve the resistance of pigs to E.coli infection.2.To investigate the key functional genes targeted and regulated by WTAP,this study used RNA-seq technology to analyze the transcriptome sequencing of PEC-J2 cells before and after WTAP interference group（siWTAP,siCtrl）and before and after E.coli infection group（E.coli,Control）.The results showed that 323 differentially expressed genes（DEGs）were screened between siWTAP and siCtrl groups,211 differentially expressed genes were screened between E.coli-infected and control groups,and 25 common DEGs existed between the two groups;KEGG enrichment analysis showed that after WTAP interference,DEGs were mainly enriched in signaling pathways such as promoting IgA intestinal immunity,antigen processing and presentation,glycosphingolipid biosynthesis,and CAMs signaling.After E.coli infection,DEGs were mainly enriched in Wnt signaling pathway,cellular complement signaling pathway,Glycosphingolipid biosynthesis pathway,etc.3.To investigate the mechanism of WATP-mediated m6A modification on the regulation of GCNT2,this study first used bioinformatics software to predict the m6A modification sites of GCNT2 gene,and found that there were two potential m6A modification sites（m6A-3294,m6A-3467）Secondly,it was verified by MeRIP-qPCR experiment that there was no significant difference in the modification level of GCNT2 gene m6A-3467 between the WTAP interference group and the control group（P>0.05）,while the modification level of m6A-3294 in the WTAP interference group was significantly decreased.（P<0.01）;The wild-type（WT）and mutant vectors（Mut）of GCNT2 m6A modification site,WTAP interference（siWTAP）and control vector（siCtrl）,YTHDF2 interference（siYTHDF2）and control vector（siCtrl）were constructed,respectively.Dual luciferase activity assay revealed that GCNT2 activity in the GCNT2-WT group was highly significantly different between the siWTAP and siCtrl groups,siYTHDF2 and siCtrl（P<0.01）,while GCNT2 activity in the GCNT2Mut group was not significantly different between the treatment groups（P>0.05）.RIP-qPCR validation showed a significant binding relationship between YTHDF2 protein and GCNT2.The YTHDF2 interference and overexpression recombinant plasmids were constructed and transfected into IPEC-J2 cells.The YTHDF2 interfering and overexpressing vector s were constructed.qRT-PCR and Western blot were used to verify the efficiency of interference and overexpression,and it was found that both GCNT2 mRNA and protein expression levels were highly significantly increased after YTHDF2 interference,while GCNT2 expression was highly significantly decreased after YTHDF2 overexpression（P<0.01）,and downregulation trend after the addition of OE-YTHDF2 in the siWTAP group.Actinomycin D treated IPEC-J2 cells in OE-YTHDF2 and OE-Ctrl groups at different time points（0h,3h,6h）,respectively.qRT-PCR detection showed that the expression of OEYTHDF2 mRNA decreased more obviously.showed that YTHDF2 affects the mRNA stability of GCNT2.Functional verification showed that the number of E.coli F18 adherent cells was significantly reduced after GCNT2 interference,and the expression of ABO,FUT1,FUT2,and FUT2A,genes related to the sphingolipid biosynthesis pathway,were significantly downregulated（P<0.01）.This indicates that WTAPmediated m6A modification has a regulatory effect on GCNT2 gene expression,and GCNT2 participates in the glycosphingolipid biosynthesis pathway and plays an important role in the process of E.coli F18 infection of porcine intestinal epithelial cells.Based on the above research results,WTAP may rely on the m6A recognition protein YTHDF2 to target and regulate the GCNT2 gene and reduce the expression level of GCNT2 gene by affecting its mRNA stability.Then inhibit the receptor formation-related signaling pathway in E coli-glycosphingolipid biosynthesis and helps IPEC-J2 to resist the infection of E.coli F18 to a certain extent ultimately. |
PDF Full Text Request