Effects And Mechanism Of CircDCP2/ssc-miR-23b/IRF1 In The Injury Of IPEC-J2 Cells Induced By CPB2 Toxin

Diarrhea is a leading cause of piglet death,and results in huge economic losses to the pig husbandry.Clostridium perfringens type C(C.perfringens type C)produces?and?(1,2)toxins,which is an important cause of diarrhea in newborn piglets.C.perfringens beta2(CPB2)produced by this organism is considered to be an important virulence factor for necrotizing enteritis in piglets.Micro RNA(miRNA)and circular RNA(circ RNA)play an important regulatory role in physiological and pathological processes such as cell metabolism and pathogenic microbial infections.It was found that CPB2 had toxic effects on some human cell lines,which could reduce the cell viability and induce different degrees of toxicity in a cell type dependent manner.However,whether CPB2 toxin can induce the injury of piglet intestinal epithelial cells,as well as the changes and regulatory mechanisms of miRNA and circ RNA during this process have not yet been reported.In this study,the CPB2 toxin protein was obtained by genetic recombination,and the CPB2 toxin-induced porcine jejunal mucosal epithelial cell(IPEC-J2)injury model was constructed.RNA sequencing(RNA-seq)technology was used to detect the changes in the expression profiles of miRNA and circ RNA in IPEC-J2 cells after CPB2 treatment,and to predict,screen and construct a circ RNA-miRNA-m RNA ce RNA regulatory network.Using quantitative real time polymerase chain reaction(RT-q PCR),fluorescence in situ hybridization(FISH),western blot(WB),dual luciferase reporter gene,flow cytometry and other methods to study the regulation and mechanism of miRNA and circ RNA in the process of CPB2-induced IPEC-J2 cell damage.The results of the research are as follows:(1)CPB2 toxin protein with biological activity was obtained by recombinant expression.After IPEC-J2 cells were treated with 10,20,30,40 and 50?g/m L CPB2 toxin,cell viability decreased in a dose-dependent manner(p<0.05),and lactate dehydrogenase(LDH)content increased in a dose-dependent manner(p<0.05).When the concentration of CPB2 was 20?g/m L,the activity of IPEC-J2 cells was about 50%.With the increase of CPB2 concentration(0,10,20,30?g/m L),the expression of TNF-?and IL-8 in IPEC-J2 cells gradually increased(p<0.05),while the expression of IL-10 gradually decreased(p<0.05).In addition,CPB2treatment of IPEC-J2 cells increased the apoptotic rate,ROS content,and Bax protein expression(p<0.05),and decreased mitochondrial membrane potential and Bcl-2 and Bcl-x L protein expression(p<0.05).The expression of ZO-1,CLDN12 and occludin OCLN was down-regulated in CPB2-treated IPEC-J2 cells(p<0.05).(2)The RNA-seq technology was used to detect the change of miRNA expression profile during CPB2 induced IPEC-J2 cell injury.Under the conditions of|log₂FC|>1 and p<0.05,419 differentially expressed miRNAs were screened,of which 227 were up-regulated,and 192 were down-regulated.The RT-q PCR results of randomly selected 8 up-regulated and8 down-regulated differentially expressed miRNAs showed consistent trends with the results of RNA-seq.Through target gene prediction,13618 target genes were obtained,and 563differentially expressed target genes were obtained after crossing with 721 differentially expressed m RNA obtained by m RNA sequencing.The function of the differentially expressed target genes GO are mainly enriched in biological processes such as the immune system process,response to stimulus,cell killing,and detoxification.Enrichment analysis of KEGG signaling pathway shows that differentially expressed target genes are mainly enriched in the TNF signaling pathway,cytokine-cytokine receptor interaction are related to immune response and inflammatory response signal pathway.The RT-q PCR results of 8 up-regulated and 8 down-regulated differentially expressed miRNAs showed consistent trends with the sequencing results.(3)The RNA-seq technology was used to detect the change of circ RNA expression profile during CPB2 induced IPEC-J2 cell damage.Under the condition of|log₂FC|>1 and p<0.05,63 differentially expressed circ RNAs were screened,of which 33 circ RNAs were up-regulated and 30 circ RNAs were down-regulated.Sanger sequencing was performed on 10differentially expressed circ RNAs,and the sequence information of the circularization sites obtained was consistent with the RNA-seq sequencing results.The RT-q PCR results of the10 differentially expressed circ RNAs showed the same trend as the RNA-seq results.Through the integration analysis of circ RNA,miRNA and m RNA,103 circ RNA-miRNA-m RNA relationship pairs were obtained.According to the above results,target genes related to the immune system,inflammatory response,and cell proliferation and apoptosis were further screened,and 20 pairs of candidate relationship pairs were obtained.Since circDCP2 has a large differential expression multiple,and IRF1 is enriched in the TNF signaling pathway,and is closely related to pathogenic microorganism infection and inflammation,circDCP2-ssc-miR-23b-IRF1 was selected for functional verification.(4)Through identification,it was found that circDCP2 is a circular RNA mainly present in the cytoplasm of IPEC-J2.circDCP2 was up-regulated in CPB2-induced IPEC-J2cells(p<0.05).circDCP2 can increase LDH content(p<0.05),inflammatory factor level(p<0.05),apoptosis rate(p<0.05),ROS content(p<0.05),and Bax expression level(p<0.05),while reducing cell viability(p<0.05),mitochondrial membrane potential(p<0.05)and Bcl-2 expression level(p<0.05)in IPEC-J2 cells treated with CPB2.circDCP2 can negatively regulate its expression through the sponge ssc-miR-23b.ssc-miR-23b can reduce cell LDH content(p<0.05),inflammatory factor expression(p<0.05),apoptosis rate(p<0.05),and ROS content(p<0.05)and Bax expression level(p<0.05),at the same time by increasing cell viability(p<0.05),mitochondrial membrane potential(p<0.05),and Bcl-2 expression level(p<0.05)to alleviate IPEC-J2 cell injury caused by CPB2.In addition,IRF1 was identified as the direct target of ssc-miR-23b.Through rescue experiments,it was found that overexpression of ssc-miR-23b can alleviate the aggravation of CPB2-induced IPEC-J2 cell damage caused by overexpression of circDCP2.Overexpression of IRF1 can restore the alleviation of circDCP2 knockdown on CPB2-induced IPEC-J2 cell damage.The role of circDCP2 in IPEC-J2 cell injury model induced by CPB2 is to intensify the inflammatory response,inhibit proliferation and promote apoptosis through ssc-miR-23b/IRF1 axis.In this study,a CPB2-induced IPEC-J2 cell injury model was successfully constructed.Using RNA-seq,419 differentially expressed miRNAs and 63 differentially expressed circ RNAs were screened in the process of CPB2 induced IPEC-J2 cell damage.circ RNA,miRNA and target genes were used to construct a ce RNA network,and the corresponding relationship pairs related to target genes related to immune system,inflammation and cell proliferation and apoptosis were screened,20 pairs of candidate circ RNA-miRNA-m RNA relationship pairs were obtained.Because IRF1 is enriched in the TNF signaling pathway and closely related to pathogen infection and inflammatory response,and the differential expression of circDCP2 is relatively large,circDCP2-ssc-miR-23b-IRF1 was selected for functional verification.A series of experiments have been conducted to find that the role of circDCP2 in IPEC-J2 cell injury model induced by CPB2 is to intensify the inflammatory response,inhibit cell proliferation and promote cell apoptosis through ssc-mir-23b/IRF1 axis.The results provide a theoretical basis for further revealing the pathogenic mechanism of C.perfringens type C.