| Piglet bacterial diarrhea is the most common type of intestinal inflammatory disease in intensive and large-scale pig production,causing extremely serious economic losses to livestock breeding industry.Escherichia coli F18(E.coli F18)is the main pathogeny causing bacterial diarrhea of weaned piglets,and the regulation mechanism of domestic native pig breeds on its resistance to E.coli will wait to be elucidated systematically.A key candidate lncRNA-lncRNA-FUT3as about the regulation of E.coli F18 infection was screened out by omics sequencing in the early stage.Based on this,this study focused on the function and regulatory mechanism of lncRNA-FUT3as and conducted an in-depth analysis.Firstly,the relationship between lncRNA-FUT3as expression level and E.coli F18 resistance was verified systematically.Secondly,RNA pull down,RIP-qPCR,ChIP-qPCR and Western blot were used to explore the molecular regulation mechanism of lncRNA-FUT3as on target gene.Then,RNA interference,overexpression,CRISPR/Cas9 knockout technique and a series of experiments were performed to verify the relationship between the expression level of target gene and E.coli F18 resistance.Finally,the interaction proteins and downstream regulation mechanisms of target gene were explored using transcriptome sequencing,His pull down-MS,Co IP-MS,and expression verification.This research will reveal the epigenetic regulation mechanism of lncRNA resistance to E.coli F18,and further clarify the resistance functional genes,and it will lay the foundation for formulating prevention and control strategies of bacterial diarrhea in piglets and disease-resistant breeding in the future.The main findings are as follows:1.Functional study on the relationship between lncRNA-FUT3as expression level and E.coli resistanceFirstly,qPCR and Northern blot were used to detect the differential expression of lncRNA-FUT3as between the duodenal tissues of E.coli F18-resistant and sensitive weaned piglets in Meishan piglets and Sutai piglets,and the results showed that the expression level of sensitive piglets in two pig breeds was significantly higher than that of resistant piglets(P<0.01);After E.coli F18 infection and LPS induction,its expression level also increased significantly(P<0.01);the porcine small intestinal epithelial cell line(IPEC-J2)with lncRNA-FUT3as silencing was successfully established,and the interference efficiency reached 68%;The bacteria count,gram staining,scanning electron microscopy,indirect immunofluorescence and a series of detection methods were performed,and it found that silencing lncRNA-FUT3as can significantly reduce the adhesion of E.coli F18 to IPEC-J2 cells.This indicated that lncRNA-FUT3as may play an important resistance-regulation role in the process of weaned piglets against E.coli F18 infection,and the down-regulation of its expression level will reduce the adhesion ability of small intestinal epithelial cells to E.coli F18,which will be conducive to improving the resistance of piglets to E.coli F18 infection.2.The mechanism study of lncRNA-FUT3as-mediated histone H4 modification on the expression regulation of target gene FUT3Firstly,qPCR and Western blot detection showed that silencing lncRNA-FUT3as could significantly reduce the mRNA and protein expression levels of target gene FUT3(P<0.01);Secondly,it was found that lncRNA-FUT3as combined with Histone H4 by RNA pull down test,and verified by Western blot and RNA immunoprecipitation(RIP)that lncRNA-FUT3as and Histone H4 existed interactions;ChIP-qPCR test was used to find that Histone H4 can be extremely significantly enriched in the FUT3 promoter region(P<0.01),and Histone H4 had a significant binding effect on the promoter region of target gene FUT3;the effect of lncRNA-FUT3as on the levels of Histone H4 acetylation(H4K16ac,H4K12ac,H4K8ac,and H4K5ac)was analyzed by Western blot,and the results showed that H4K16ac and H4K8ac protein expression was decreased after silencing lncRNA-FUT3as,and ChIP-qPCR verification found that lncRNA-FUT3as could significantly reduce the H4K16ac acetylation level in the FUT3 promoter region(P<0.05);the normal IPEC-J2 cells and siRNA interference cell lines were treated with acetylation inhibitors(TSA and SSAHA),and the results showed that after TSA and SSAHA treatmenting,the mRNA expression level of FUT3 gene was extremely significant decreased(P<0.01),and the protein expression of FUT3 was also showed a significant decrease by Western blot detection.This indicated that lncRNA-FUT3as can mediate histone H4 acetylation modification(H4K16ac)to regulate the expression level of target gene FUT3.3.Functional study on the relationship between the expression level of target gene FUT3 and E.coli F18 resistanceFirstly,the expression profile of different tissues in 35-day-old weaned piglets was examined,and found that FUT3 gene was expressed in different tissues,especially highly expressed in duodenum and jejunum.QPCR was used to detect the differential expression of FUT3 in the duodenum and jejunum tissues of E.coli-resistant and sensitive weaned piglets,and the results showed that the expression level in intestinal tissue of sensitive piglets was significantly higher than that of resistant piglets(P<0.01);Immunohistochemical(IHC)analysis showed that FUT3 gene is mainly distributed on the epithelial mucosal surface of small intestine tissue,and sensitive individuals are significantly higher than resistant individuals;After LPS inducion and bacterial infection of E.coli F18,the mRNA and protein expression levels of FUT3 showed significant upregulation(P<0.05);the IPEC-J2 cell lines with FUT3 siRNA interference,overexpression,and CRISPR/Cas9 knockout were successfully constructed,and FUT3 expression level can significantly affect the adhesion ability of E.coli F18 to IPEC-J2 cells,which were found by bacteria count,gram staining,and indirect immunofluorescence observation,respectively.This indicated that the target gene FUT3 can play an important resistance-regulation role in the process of weaned piglets against E.coli F18 diarrhea,and the downregulated of its expression level will reduce the adhesion ability of small intestinal epithelial cells to E.coli F18,which will be conducive to improving resistance of piglets to E.coli F184.Analysis of downstream regulatory mechanisms of lncRNA-FUT3as targeting FUT3The transcriptome sequencing analysis of IPEC-J2 cells before and after lncRNA-FUT3as interference was performed,and bioinformatics was used to screen for differentially expressed genes and the enriched signal pathways.The results showed that there were 388 differentially expressed genes between lncRNA-FUT3as interference group and the control group,of which 199 genes were downregulated in the interference group;The KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly concentrated in 20 signal pathways;combining with the analysis results of the previous sequencing data,one important signal pathway—Glycosphingolipid biosynthesis-lacto and neolacto series(ko00601)was screened out,and some signaling pathway genes(FUT2,ST3GAL3 and B3GALNT1)were also selected.After lncRNA-FUT3as interference,the mRNA and protein expression levels of FUT2,ST3GAL3 and B3GALNT1 genes decreased significantly(P<0.01);After overexpressing the target gene FUT3,the expression levels of FUT2,B3GALNT1,ST3GAL3,and FUT4 increased significantly(P<0.01),and when FUT3 interfered or knocked out,the expression levels were depressed in the wake of FUT3 expression.QPCR was used to detect the expression changes of glycosphingolipid signaling pathway genes(FUT2,B3GALNT1,ST3GAL3,FUT4)on duodenal tissue of E.coli resistant and sensitive weaned piglets(Meishan and Sutai piglets),LPS-inducted and bacterial-stimulated IPEC-J2 cells.The results showed that FUT2,B3GALNT1,ST3GAL3,and FUT4 genes were significantly upregulated after F18ab and F18ac infection(P<0.01);After 8 h of LPS induction,both FUT4 and ST3GAL3 genes appeared significant upregulation,and the expression levels of the four detected genes in the duodenum of sensitive group were significantly higher than those in the resistant group.Fourteen interacting proteins of FUT3 were identified by using CoIP-MS and His pull down-MS detection,and none of these proteins belonged to the glycosphingolipid signaling pathway,and it was speculated that FUT3 gene may indirectly regulate glycosphingolipid biosynthesis.This indicated that FUT3 may affect piglets’ susceptibility to E.coli F18 by indirectly regulating the glycosphingolipid biosynthesis signal pathway.Based on the above studies,it is shown that the antisense long non-coding RNA-lncRNA-FUT3as may regulate the expression level of the target gene FUT3 by mediating the acetylation of Histone H4K16ac,and then affect the piglet’ susceptibility to Escherichia coli F18 by activating/inhibiting the glycosphingolipid biosynthetic pathway. |
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