Regulatory Mechanism Analysis Of MUC2 And TFF1 Genes In Porcine Epidemic Diarrhea

Porcine epidemic diarrhea is an infectious disease caused by porcine epidemic diarrhea virus with high infectivity and mortality,which easily contributes to major economic losses in large pig farms.Severe diarrhoea,diarrhea,and dehydration are the clinical manifestations.Pigs are susceptible to PEDV at all ages,and the younger the age,especially the higher the morbidity and mortality of suckling piglets.Drugs and vaccines are currently unable to fully treat and prevent the incidence of PEDV.Impaired intestinal mucosal integrity is closely linked to disease.MUC2 and TFF1 are gastrointestinal secreted proteins which repair of gastrointestinal mucous membranes and play an important role in the defense.They may form a protective layer at the gastrointestinal epithelium surface,protect and lubricate the epithelium surface and preserve the epithelium’s integrity.These can also help cells bind and provide protection against bacteria,toxins,and foreign bodies,as well as contribute to the creation of intestinal mucus barriers.The aim of this research,based on the biological role of MUC2 and TFF1,was to examine the regulatory effects of MUC2 and TFF1 on resistance to PEDV infection and to investigate the effect of DNA methylation in the promoter region on the expression of MUC2 and TFF1 genes.The main results were as follows:1.In this study,changes were observed in intestinal mucosa of piglets after infection with PEDV.At the same time,the expression of MUC2 and TFF1 genes at the tissue and cell level were detected,and RNA interference at the cell level for MUC2 and TFF1 on PEDV replication influences was examined.The results of paraffin section showed that the intestinal villi of porcine epidemic diarrhea piglets were damaged and shed,the height and density of intestinal villi were decreased,and the lamina propria was exposed,while the normal piglet’s intestinal mucosal barrier structure was complete and hierarchical.The results of qRT-PCR indicated that the expression of MUC2 gene in the small intestine(duodenum,jejunum,and ileum)in the diarrhea group was significantly higher than that in the normal group(P<0.01).There was no difference in the expression of TFF1 in the duodenum,while in the jejunum and ileum,the expression of TFF1 in the diarrhea group was significantly higher than that in the normal group(P<0.01).Furthermore,we infected IPEC-J2 cells with PEDV(0.1 MOI)to detect changes in expression of MUC2 and TFF1 at different times.The results indicated that the expression of MUC2 gene was significantly up-regulated at 24h of infection(P<0.05).The expression of TFF1 gene was first decreased and then increased,and it was significantly up-regulated at 48h of infection(P<0.01).Additionally,RNAi was used to suppress the expression of MUC2 and TFF1,and found that the expression of M gene in the genome of the PEDV had increased significantly(P<0.01).These results provided indirect evidence that high expression of MUC2 and TFF1 is conducive to the resistance of piglets to PEDV infection.2.Bisulfite sequencing PCR(BSP)was used to detect the degree of DNA methylation in the MUC2 and TFF1 promoter regions,and the relationship between methylation in the promoter region of MUC2 and TFF1 genes and mRNA expression was analyzed.To detect the effect of methylation status on promoter function,the dual-luciferase reporter gene system was employed.The findings showed a negative correlation between the DNA methylation in promoters and the expression of the MUC2 and TFF1 genes,and the methylation of MUC2 mC-5 and TFF1 mC-6 sites had a significant negative association with the expression of mRNA(P<0.05).Moreover,DNA methylation significantly decreased MUC2 and TFF1 promoter luciferase activity(P<0.01)was observed by the dual-luciferase reporter gene system.3.The potential transcription factor binding sites in the promoter region of MUC2 and TFF1 were predicted,and it was found that the MUC2 mC-5 and TFF1 mC-6 sites were located in the transcription factor YY1 and C/EBPa binding domains,respectively.The binding of transcription factor YY1 to MUC2 promoter sequences and C/EBPα to TFF1 promoter sequences were verified by ChIP assay.After interfering with the transcription factor YY1 and C/EBPa,the expression of MUC2 and TFF1 genes were significantly decreased(P<0.05).Therefore,it is speculated that the methylation of MUC2 mC-5 and TFF1 mC-6 sites has been shown to interfere with the binding of transcription factors and further inhibit the expression of MUC2 and TFF1 genes.In summary,the intestinal mucosa of the diarrhea group was damaged and the mucus barrier was destroyed.Then the expression trend of MUC2 and TFF1 was higher than that of normal piglets.And the methylation of the MUC2 mC-5 and TFF1 mC-6 sites may hinder the binding to the transcription factors,inhibit the expression of MUC2 and TFF1 genes,thereby increasing the susceptibility of piglets to PEDV.This study reveals the molecular regulation mechanism of DNA methylation on piglets against PEDV infection from the epigenetic level,which provides a theoretical framework for the study of PEDV’s pathogenesis from an intestinal mucosal barrier perspective.