Regulation Mechanism Of Long Noncoding RNA (LncRNA) And MRNA On Resisting To Piglet Diarrhea Caused By Clostridium Perfringens Type C

Piglet diarrhea is a common intestinal disease,which seriously affects the economic efficiency of swine industry.Clostridium perfringens(C.perfringens)type C is one of the bacterial pathogens causing piglet diarrhea,it can adhere to the epithelial cells of intestines in piglets,and releases a variety of toxins,damaging intestinal villi,mucosa and epithelial cells.Though the vaccine prevention and antibiotic treatment decrease and control the occurrence of piglet diarrhea caused by C.perfringens type C,the long-time use of antibiotics leads to the increasing resistance of pathogens and bring the great risks to food safety and human health,meanwhile,the variation of pathogen constantly set a high standard for vaccine development,which make the prevention and treatment of piglet diarrhea more and more difficult.Long noncoding RNA(lncRNA)is a type of transcripts with length of more than 200 nucleotides and without protein coding potential,it can activate or inhibit the expression levels of protein coding genes and plays important regulatory functions in the process of host defense against pathogen infection.Recently,studies have reported that the expressions of lncRNA and mRNA are closely associated with piglet diarrhea caused by Escherichia coli and Salmonella infection,while the regulatory mechanisms of lncRNA and mRNA in piglet response to diarrhea disease caused by C.perfringens type C are still unclear.Exploring mechanisms of lncRNA and mRNA in regulating piglet diarrhea caused by C.perfringens type C,will have great significance for improving the resistance of piglet diarrhea and breeding diarrhea-resistant pig strains.Therefore,to explore the regulatory mechanisms of lncRNA and mRNA in piglet diarrhea resistance caused by C.perfringens type C infection,the thirty 7-day-old piglets were selected as the experimental animals.In which,twenty-five piglets were randomly selected to orally inoculate with the C.perfringens type C medium to filtrate resistance(IR)and susceptibility(IS)piglets by body weight,diarrhea scores and numbers of fecal C.perfringens type C.The RNA-seq and bioinformation methods were used to obtain the differentially expressed profiles of lncRNA and mRNA of fifteen piglets from IR,IS and control(IC)groups,perform GO and KEGG functional enrichment analyses of the differentially expressed lncRNA and mRNA and screen the diarrhea resistance-associated genes.The main results are as follows:1.The infection of C.perfringens type C had significant effect on body weight,diarrhea scores and numbers of C.perfringens type C in feces,which were significantly difference in piglets between IR and IS groups.Compared to IC groups,the diarrhea scores and fecal bacterial numbers of piglets in the IR and IS groups were both increased,while the weights of heart,live,spleen and body were decreased.Compared to IS groups,the diarrhea score and fecal bacterial numbers of piglets in the IR group were significantly lower(P < 0.05),while the weights of heart,liver,spleen and body of piglets were significantly higher(P < 0.05).2.Results of expression profiles of ileum lncRNA and mRNA for piglets in the IR,IS and IC groups showed that: A total of 3,640 lncRNA(1,850 novel lncRNA and 1,890 annotated lncRNA)and 25,491 mRNA were identified among IR,IS and IC groups.These lncRNA have the lower exon number,shorter length,lower expression levels and lower conservation than mRNA.Through differential expression analyses,a total of 480 differentially expressed lncRNA and 3,669 differentially expressed mRNA were screened among IR,IS and IC groups.in which,4 lncRNA and 28 mRNA were common differentially expressed among three groups.3.Results of functional enrichment analyses showed as follows: Compared to IC group,the differentially expressed lncRNA and mRNA identified in the IR and IS groups were enriched in the similar GO functions and KEGG pathways.The enriched GO functions mainly included cell function,transduction,receptor activation,amino acid metabolism,biosynthesis,metabolism and so on.The KEGG pathways were mainly enriched in several metabolism-,biological synthesis-and signal transduction-related signaling pathways,such as oxidative phosphorylation,ABC transporters,fatty acid metabolism,graft-versus-host disease and so on.4.A total of 212 differentially expressed lncRNA corresponding to 505 differentially expressed mRNA were screened,the formed lncRNA-mRNA regulatory gene pairs may have an important function in regulating C.perfringens type C infectious disease.5.Functional prediction and analysis of differentially expressed lncRNA in IRvsIS group showed that: The target genes of differentially expressed lncRNA in IRvsIS group were mainly enriched in GO functions of MHC class I protein complex,immune response,antigen processing and presentation,regulation of I-kappa B kinase/NF-kappa B signaling.The enriched KEGG pathways included various immune-related pathways,such as cell adhesion molecules,inflammatory bowel disease,T cell receptor signaling pathway,natural killer cell mediated cytotoxicity,cytokine-cytokine receptor interaction.6.Results of qPCR detection indicated that: The expression levels of IRAK3,LCP2 and TLR8 in the IR and IS groups were extremely significantly lower than those in the IC group(P < 0.01),and the expression levels of TNFRSF11 A and CYP1A1 in the IS group were extremely significantly higher than those in the IC group(P < 0.01).The expression levels of TLR8 and CYP1A1 were significantly difference between IR and IS groups(P < 0.05).The expression levels of cytokines NF-κB and IFN-α in the IR and IS groups were also significantly higher than those in the IC group(P < 0.05),and they were significantly difference between IR and IS groups,the expression levels of IL-1β and TNF-α were significantly higher than those in the IC group(P < 0.05).In conclusion,lncRNA can regulate the expressions of their target genes,then indirectly affect levels of cytokines,and regulate the diarrhea disease of piglets in response to C.perfringens type C infection.This study can provide important foundation for regulation mechanism of piglet diarrhea caused by C.perfringens type C and the breeding of diarrhea-resistant pig strains.