Sequencing And Characterization Of LncRNA,mRNA And MiRNA In The Spleen Of Piglet Diarrhea Caused By Clostridium Perfringens Type C

Diarrhea is a commonly infectious intestinal disease,which has led to piglet death.This disease has caused great economic losses to pig industry and also has become a stumbling block to the development of pig industry.Clostridium perfringens(C.perfringens)type C is one of the important pathogenic bacteria leading to bacterial diarrhea in piglets.C.perfringens type C,mainly infected piglets by digestive tract,can quickly grow in the intestine of piglets.An increasing the number of C.perfringens type C damage intestinal tissue and then fatal toxins produced by C.perfringens type C are absorbed in the intestinal tract.Fatal toxins are transported to the terminal organs(such as spleen)by the body fluid circulation system,thus causing diarrhea and systemic organs damage in piglets.To some degree,use of vaccine and antibiotics can decrease and control the occurrence of C.perfringens type C-induced diarrhea.In addition,the antibiotics removal and green healthy feeding will become one of the directions of future development in breeding industry.Thus,it need to alleviate the environmental pollution caused by drugs and the dependence of pigs on vaccines and drugs.Studies have found that the splenic lncRNA(long non-coding RNA),mRNA and miRNA(microRNA)play important roles in piglet diarrhea caused by Escherichia coli and Salmonella.Nevertheless,the splenic lncRNA,mRNA and miRNA in regulating C.perfringens type C-induced diarrhea are still limited.Therefore,based on the regulation mechanism of piglet diarrhea,we can identify several molecules that resistance to C.perfringens type C infection,which could help us to breed a pig strain resisting to C.perfringens type C.In our study,thirty healthy 7-day-old piglets were selected as experimental animal.Among these piglets,five piglets were randomly selected as control group(SC).The other 25 piglets were orally challenged with the C.perfringens type C and were ranked from low to high based on total diarrhea scores.Then,the top five and bottom five piglets were considered as the resistant group(SR)and susceptible group(SS),respectively.Furthermore,the spleens from SC,SR and SS piglets were investigated using RNA-Seq and then bioinformatics analyses were conducted to obtain the differentially expressed lncRNA,mRNA and miRNA in three groups.These differentially expressed molecules were performed GO term and KEGG signaling pathway.We systematically studied important regulatory pathways and key molecules,which provide a reference to the regulation mechanism of lncRNA,mRNA and miRNA in C.perfringens type C-caused diarrhea.The main findings were as follows:(1)We obtained 184539 transcripts for all sequencing libraries.A total of 2056 novel lncRNA(including 1811 lincRNA and 245 antisense_lncRNA)was identified in the spleen from SC,SS and SR group.In addition,a total of 2144 annotated lncRNA and 4324 novel mRNA were screened,respectively.(2)In this study,novel lncRNA identified had similar genomic features with the annotated lncRNA in ALDB(A Domestic-Animal Long Noncoding RNA)database.However,compared to the protein-coding genes,novel lncRNA identified in this study had fewer the number of the exon,longer transcript length,shorter ORF(open reading frame),much lower expression level,lower protein coding potency and less conservative sequence.(3)miRNA-Seq yielded a total of 338 known mature miRNA in the spleen,corresponding to 295 pre-miRNA.Moreover,a total of 514 novel mature miRNA was identified,corresponding to 257 pre-miRNA.(4)Differentially expressed lncRNA,mRNA and miRNA in the spleen of piglets in SC,SS and SR groups were performed using hierarchical clustering maps method.The result showed that the SR and SS were clustered together and suggested that two groups had similar expression pattern.(5)A total of 19 differentially expressed lncRNA(including 9 up-regulated and 10 down-regulated lncRNA)was screened from the C.perfringens type C-resistance piglets and the C.perfringens type C-susceptibility piglets.We performed the target genes prediction of these differentially expressed lncRNA based on cis and trans mechanism.Target prediction indicated that 23 target genes within the downstream and upstream of these differentially expressed lncRNA were predicted based on cis mechanism.In addition,a total of 93 target genes had significant correlation with these differentially expressed lncRNA based on trans mechanism.The functional enrichment analysis of GO term showed that these target genes were mainly participated in NK T cell activation,Regulation of type 2 immune response and Defense response to bacterium.The enriched KEGG signaling pathways included Regulation of autophagy,Influenza A,Inflammatory bowel disease.Our results indicated these GO terms and KEGG signaling pathways were associated with diarrhea caused by C.perfringens type C.(6)A total of 123 differentially expressed genes(including 43 up-regulated and 80 down-regulated gene)were screened from the C.perfringens type C-resistance and the C.perfringens type C-susceptibility individuals.GO terms enrichment showed that these differentially expressed genes were mainly involved in Immune system process,Defense response,Innate immune response and Response to virus;KEGG pathway enrichment showed that these differentially expressed genes mainly included Amoebiasis,Influenza A,Measles,Legionellosis,and NF-kappa B signaling pathway.These GO terms and KEGG signaling pathways were possibly related to the resistance and susceptibility to C.perfringens type C in piglets.(7)A total of 56 differentially expressed miRNA(including 21 up-regulated and 35 down-regulated miRNA)was screened from the C.perfringens type C-resistance and the C.perfringens type C-susceptibility individuals.The enriched GO terms of target genes of these differentially expressed miRNA were mainly involved in Immature T cell proliferation and Regulation of defense response.These results suggested that differentially expressed miRNA targeting their genes may play key roles in regulating the infection to C.perfringens type C in piglets.(8)Combing with target prediction and function analyses,we identified two important lncRNA(ALDBSSCT0000006918 and ALDBSSCT0000007366),which were related to C.perfringens type C infection.Two lncRNA all belonging to lincRNA subtype were located on pig chromosome 3.The enriched KEGG signaling pathways of two lncRNA target genes(such as GADD45 G and IL12A)mainly included MAPK and Toll like receptor signaling pathway,which revealed that differentially expressed ALDBSSCT0000006918 and ALDBSSCT0000007366 regulated their target gene to involve in immune-related pathways to C.perfringens type C infection.(9)Based on target gene prediction and functional analyses,we screened four miRNA(including miR-133 b,miR-532-3p,miR-339-5p and miR-331-3p),which were associated with resistance to C.perfringens type C infection.The downregulated miR-113 b and up-regulated miR-532-3p in SR targeted NFATC4.The upregulated miR-339-5p and miR-331-3p in SS targeted TNFAIP8L2.Additionally,miR-339-5p also targeted HTRA3.These target genes influenced the expression of downstream immune genes and cytokine genes.These resistant miRNA may regulate the immune response to C.perfringens type C and will regarded as the key target of resistance of piglets to this pathogen.In conclusion,the differentially expressed lncRNA,mRNA and miRNA in the spleen of C.perfringens type C-resistance and C.perfringens type C-susceptibility piglets were explored.We identified two key lncRNA(ALDBSSCT0006918 and ALDBSSSCT0000007366)and four important miRNA(miR-133 b,miR-532-3p,miR-339-5p and miR-331-3p)by bioinformatics prediction and functional analyses,which were associated with the biological process of C.perfringens type C infection in piglets.In the future,it is necessary to further verify their specific regulatory mechanism at the cellular level.To some degree,our study provided theoretical foundation for breeding a pig strain resisting to C.perfringens type C.