Transcriptome And MicroRNAome Analysis Reveals Molecular Mechanism Of Intestinal Epithelial Crypt-villus Axis Renewal In Weaned Piglets

Early weaning stress syndrome in piglets is an important event in the large-scale breeding of pigs,causing huge economic losses to the agriculture enterprises every year.Evidences have proved that weaning can lead to atrophy of piglet intestinal villi,digestive and absorption disorder,and inflammation,which induce the release of a large number of reactive oxygen species free radicals;eventually causing intestinal dysfunction and diseases.Our previous study found that the antioxidant capacity of intestinal epithelial cells(IECs)of weaned piglets gradually decreased with differentiation;and the proteomic analysis revealed that the differential expression proteins of crypt-villus axis are mainly involved in carbon metabolism,amino acid and protein metabolism,and oxidative phosphorylation,etc.;indicates that energy metabolism plays an key role in the IECs renewal.In order to explore the molecular mechanism of intestinal epithelial crypt-villi axis renewal in early weaned piglets,the present study was performed on transcriptome and sRNA high-throughput sequencing of the intestinal epithelial villus upper cells(F1)and crypt cells(F3)of the jejunum at 21 days of suckling(day 0 for weaning)and days 1,3,7 and 14 after weaning.The specific research results are as follows:(1)Transcriptome sequencing analysis showed that there were 31,77,154 and 112 differentially expressed genes(DEGs)in F1 at different ages(1,3,7 and 14 days)after weaning,compare with F1 of 21-day-old suckling piglets.GO analysis showed that the functions of DEGs are mainly involved in biological processes such as metabolism,response to stimulation and immunity.KEGG analysis showed that DEGs are enriched in related pathways such as hormone synthesis,immunodeficiency,arachidonic acid metabolism and signal transduction.Furthermore,screening for DEGs revealed that the expression of brain-gut peptides(GHRL,GIP,SS,GLG,and MTL),Slc39a4,CYCS,and RPS19 continued to be down-regulated;while the expression of PLA2,PGFS,GST,NQO1,CYP1A1,CES,and IFITM1 continued to be up-regulated.Among them,PLA2,an important enzyme of phospholipid metabolism,increased significantly on the 3th and 7th day after weaning and had the highest differential expression multiples(P<0.01),suggesting that weaning stress may promote phospholipid metabolism of cell membrane.(2)Small RNA sequencing analysis showed that there were 246 known miRNAs and 119 new miRNAs in F1,and 78,108,131 and 161 differentially known miRNAs were detected in F1 at different ages(1,3,7 and 14 days)after weaning,compare with F1 of 21-day-old suckling piglets.Among them,expression of let-7d-3p was significantly up-regulated,while miR-100 expression was significantly decreased in the first week after weaning.Bioinformatics predicts target genes of differentially known miRNAs,and KEGG pathway analysis showed that the target genes were mainly enriched in biological processes such as Notch signaling pathway,glyceride/glycerol phospholipid metabolism,protein digestion and absorption,vitamin digestion and absorption,and fatty acid metabolism.(3)Transcriptome sequencing analysis showed that there were 32,70,189 and 102 DEGs in F3 at different ages(1,3,7 and 14 days)after weaning,compare with F3 of 21-day-old suckling piglets.GO analysis showed that the functions of DEGs are mainly involved in biological processes such as metabolism,development and response to stimulation.KEGG analysis showed that DEGs are enriched in related pathways such as hormone synthesis,energy metabolism,immunodeficiency and metabolism of exogenous compounds.Furthermore,screening for DEGs revealed the expression of brain-gut peptide(GHRL,GIP,GLG,and MTL),Slc39a4,TFF3,and CLCA1 was continuously down-regulated;while nutrient transport carriers(Slc5al2,Slc3al,Slc15al,and Slc2a2),PLA2,PGFS,CYP1A1,TRIM5 and IL-18 was continuously up-regulated.(4)Small RNA sequencing analysis showed that there were 220 known miRNAs and 95 new miRNAs in F3,and 62,69,89 and 115 differentially known miRNAs were detected in F3 at different ages(1,3,7 and 14 days)after weaning,compare with F3 of 21-day-old suckling piglets.Among them,expression of miR-9 was continued significantly up-regulated in the first week after weaning;while expression of miR-122 was significantly down-reglutaed on the first day after weaning,and then its expression level increased significantly from day 3 after weaning.Bioinformatics predicts target genes of differentially known miRNAs,and KEGG pathway analysis showed that the target genes are mainly enriched in biological process such as Notch signaling pathway,basal cell carcinoma,phosphoinositide metabolism,glyceride/glycerol phospholipid metabolism,focal adhesions,base excision repair and amino acid metabolism,etc..(5)sRNA sequencing analysis that there were 15 up-regulated and 41 down-regulated miRNAs in F3 of the jejunum compared to F1.Notably,we found that miR-100 was significantly decreased in F3,and the expression differential fold was the largest;qRT-PCR confirmed that the expression level of miR-100 in F1 was significantly higher than that of F3(P<0.01).Transfection of miR-100 mimics in IPEC-J2 cells significantly increased content of alkaline phosphatase in the cells(P<0.05).MiR-100 can inhibited cell proliferation by inducing cell cycle G0/G1 arrest.We also showed that miR-100 significantly inhibited the migration of IPEC-J2 cells and promoted cell apoptosis induce cell apoptosis via caspase-3-dependent cleavage of Bcl-2 pathway.Bioinformatics combined with the dual-luciferase reporter gene verified that FGFR3 is a target of miR-100.We confirmed that overexpression of miR-100 suppressed FGFR3 expression in IPEC-J2 cells by directly targeting the FGFR3 3'-UTR.(6)The ORF sequence of the porcine FZD6 gene(GenBank accession number:KJ808827)was 2139 bp,encoding 712 amino acid proteins with 7 transmembrane domains and a cysteine-rich domain at the extracellular N-terminus.Although FZD6 is widely expressed in various tissues,it also has certain expression differences among different tissues.The results show that in the jejunum,FZD6 protein is highly expressed in the villus and less in the crypt cells(P<0.05).Knockdown of FZD6 gene can significantly inhibited cell proliferation and promoted apoptosis of IPEC-J2 cells.Furthermore,qPCR and Western blot analysis revealed that Rac1,RhoA and JNK1,some components of Wnt/PCP signaling pathway were upregulated(P<0.05)by knockdown of FZD6 in IPEC-J2 cells.