Functional Mechanism Of Lnc001776 Modulating CPB2 Toxin-induced Injury Of IPEC-J2 Cells Via Ssc-let-7i-5p/IL-6

Piglet diarrhea is one of the common intestinal diseases in the process of large-scale pig farming,which leads to slow growth and death of piglets and seriously affects the healthy development of pig farming.Clostridium perfringens（C.perfringens）beta2（CPB2）toxin produced by C.perfringens type C damages the piglet intestine and causes necrotizing enteritis in piglets.In recent years,piglet diarrhea has been controlled to a certain extent through the use of antibiotics and vaccines,but with the emergence of problems such as food safety,environmental pollution and the call for green,antibiotic-free farming,new challenges have been brought to the control of piglet diarrhea.Therefore,studying the diarrheal mechanism of piglets’resistance to pathogenic bacterial infection from the molecular genetic perspective and essentially improving piglets’anti-diarrheal ability has become one of the important ways to prevent and control piglets’diarrhea.LncRNA and miRNA are involved in regulating intestinal diseases caused by pathogenic microbial infections.However,their functions and regulatory mechanisms in C.perfringens type C-infected diarrhea in piglets are unclear.In view of this,based on the RNA-seq data of the ileum tissue of C.perfringens type C-infected piglets in the early stage of the group,this study screened out lnc RNAs and miRNAs that were co-differentially expressed in the ileum tissue of diarrheal piglets and CPB2 toxin-treated IPEC-J2 cells to construct a lnc RNA-miRNA-m RNA regulatory network.RT-q PCR,Western Blot,ELISA,Ed U staining,flow cytometry and dual luciferase gene reporter were used to investigate the regulatory mechanisms of lnc RNA and miRNA in CPB2 toxin-induced IPEC-J2 cell injury.The main results were as follows:（1）FDR<0.05,FPKM>1 and|log₂ FC|>1 were used as screening conditions and excluded lnc RNAs without target genes,and 6 differentially expressed lnc RNAs that their target genes enriched in inflammatory and immune-related pathways were screened in the previous RNA-seq data.The expression of these 6 differentially expressed lnc RNAs in ileum tissue of diarrheic（susceptible and resistant）piglets and in CPB2-induced IPEC-J2 cells was examined by RT-q PCR.The results showed that lnc001776 had the largest differential fold in both tissues and cells and was highly significantly up-regulated in both（P<0.01）.The tissue expression profile results indicated that lnc001776 was highly expressed in duodenum,jejunum,and ileum.The full-length sequence of lnc001776 was 3386 bp by 5’and 3’RACE assays.Overexpression of lnc001776 increased pro-inflammatory cytokines levels,LDH activity and ROS levels,decreased anti-inflammatory cytokines levels,SOD activity and cell viability,inhibited cell proliferation,facilitated apoptosis,and disrupted inter-cellular tight junctions,which in turn aggravated CPB2 toxin-induced IPEC-J2 cells injury.Knockdown of lnc001776 ameliorated the damaged effect of CPB2 toxin in IPEC-J2 cells.Cytoplasm/nuclear RNA separation assay and RNA-FISH results indicated that lnc001776 was mainly localized to the IPEC-J2 cytoplasm.Differentially expressed miRNA（ssc-let-7i-5p）with binding sites and expression negatively correlated with lnc001776 were screened from previous RNA-seq data.Its target genes that were related to inflammation and immune infectious diseases were predicted and screened out,and took intersection with differential m RNAs to construct ceRNA regulatory network.Based on the research background,the regulatory axis of lnc001776/ssc-let-7i-5p/IL-6 was screened.（2）The expression of ssc-let-7i-5p was significantly down-regulated in the ileum tissue of diarrheic piglets and in CPB2 toxin-treated IPEC-J2 cells（P<0.01）,and expression was lowest at 24 h of CPB2 toxin treatment（P<0.01）.The results of tissue expression profile showed that ssc-let-7i-5p was most highly expressed in the ileum,and was also highly expressed in the spleen,jejunum,lung and liver.Overexpression of ssc-let-7i-5p had no significant effect on ROS levels and SOD viability,whereas suppressed pro-inflammatory cytokines levels,LDH activity and apoptosis,increased anti-inflammatory cytokine levels and cell viability,promoted cell proliferation and improved intestinal epithelial barrier function,thereby alleviating CPB2 toxin-induced IPEC-J2 cell injury.（3）Dual luciferase activity assays indicated that ssc-let-7i-5p was able to target binding to lnc001776 and IL-6,respectively.Overexpression of lnc001776 decreased ssc-let-7i-5p expression and increased IL-6 expression,and knockdown of lnc001776had the opposite results.In addition,overexpression of ssc-let-7i-5p inhibited the facilitative effect of lnc001776 on IL-6 levels.Rescue assays demonstrated that overexpression of ssc-let-7i-5p blocked the promoting effect of lnc001776 on CPB2toxin-induced inflammation,apoptosis and tight junction disorder in IPEC-J2 cells,while overexpression of IL-6 suppressed the blocking effect of ssc-let-7i-5p on lnc001776,thereby restoring the effect of lnc001776 on CPB2 toxin-induced damaged effects in IPEC-J2 cells.In summary,the lnc001776/ssc-let-7i-5p/IL-6 ceRNA interplay network played an important regulatory role in CPB2 toxin-induced IPEC-J2 cell injury,e.g.,lnc001776promoted CPB2 toxin-induced inflammatory response,apoptosis and intestinal barrier dysfunction in IPEC-J2 by sponging ssc-let-7i-5p to enhance the expression of IL-6,ultimately aggravating CPB2 toxin injury to IPEC-J2 cells.The results of this study may provide a theoretical basis for further revealing the mechanism of the role of lnc RNAs in C.perfringens type C-infected piglet diarrhea.