Functional Mechanism Of M~6A Modification In CPB2 Toxin-indued Injury Of Porcine IPEC-J2 Cells

Piglet diarrhea is the main cause of high mortality of piglets in large-scale breeding.Clostridium perfringens beta2 toxin（CPB2）is a key virulence factor for necrotizing enteritis in piglets.At present,the prevention and treatment of piglet diarrhea by antibiotics and vaccines is not only limited,its side effects and metabolites will bring hidden dangers to human health and living environment.Comparatively speaking,improving the anti-diarrhea ability of piglets by molecular breeding technology could fundamentally reduce the incidence of diarrhea of piglets,which is an effective means to prevent and treat diarrhea of piglets.Studies have shown that N⁶-methyladenosine（m⁶A）and m⁶A methyltransferase-like 3（METTL3）play an important role in Escherichia coli and virus-infected livestock and poultry diarrhea and regulate gene expression to resist the occurrence of diarrhea.However,the function and regulatory mechanism of m⁶A modification in piglet diarrhea caused by Clostridium perfringens type C（C.perfringens type C）remains unclear.Therefore,the functional and molecular regulation mechanism of m⁶A modification in porcine IPEC-J2 cells induced by CPB2toxin were analyzed by m⁶A-seq,RNA-seq technology,Western Blot,CCK8,LDH,RT-qPCR,ELISA,immunofluorescence,MeRIP-qPCR,half-life and double luciferase report assays.This study provides an effective molecular target for the prevention of C.perfringens diarrhea in piglets and a theoretical basis for molecular disease resistance breeding.The main results were as follows:1.The results of RT-qPCR and m⁶A methylation quantitative assay showed that m⁶A methylase（METTL3,METTL14,WTAP）and demethylase（FTO,ALKBH5）were significantly changed in ileum tissue of C.perfringens diarrhea piglets and IPEC-J2 cells injury model induced by CPB2.The expression trends of METTL3,WTAP and ALKBH5 were the same in vivo and in vitro models,and METTL3 and ALKBH5 were highly expressed in IS,IR and CPB2 groups,while WTAP was low expressed,the level of m⁶A increased significantly.Those suggest that m⁶A methylation modification played an important role in the piglet diarrhea caused by C.perfringens type C infection.2.Using m⁶A-seq,RNA-seq and bioinformatics methods,it was found that m⁶A modification widely existed in IPEC-J2 cells induced by CPB2,and was mainly distributed in 3’-UTR region,termination codon region,coding sequence（CDS）and 5’-UTR region.The m⁶A motif was mainly composed of GGACU.Using|log₂（fold change）|>0.585 and P<0.05 as the screening conditions,m⁶A-seq results showed that there were 1448 m⁶A peaks（1314 genes）with significant differences,of which 437（394 genes）were up-regulated and 1011（920 genes）were down regulated.RNA-seq results showed that there were 1636 differentially expressed genes（1094 up-regulated and 542 down regulated）.The association analysis of m⁶A-seq,RNA-seq,m⁶A-seq and RNA-seq showed that Wnt signaling pathway and related genes played a key role in IPEC-J2 cells induced by CPB2 through m⁶A methylation modification.3.The results of plasmonuclear separation and FISH assays showed that METTL3was mainly distributed in the nucleus of IPEC-J2 cells.Quantitative assay of m⁶A methylation showed that METTL3 positively regulated the m⁶A level of cells.CCK8,LDH,RT-qPCR,ELISA and immunofluorescence assays showed that METTL3inhibited CPB2-induced IPEC-J2 cell viability,aggravated cell damage,promoted inflammatory reaction,and destroyed the tight cell connection.MeRIP qPCR,half-life and double Luciferase report showed that METTL3 targeted WNT9A,increased m⁶A level of WNT9A mRNA,decreased WNT9A mRNA stability and decreased WNT9A mRNA expression.Rescue assay showed that WNT9A overexpression could reverse the damage of METTL3 to IPEC-J2 cells induced by CPB2.In conclusion,m⁶A methylation modification is involved in the pathological process of piglet diarrhea caused by Clostridium perfringens type C,in which m⁶A methylase METTL3 targeted WNT9A in the way of m⁶A methylation modification and promoted the injury response of IPEC-J2 cells induced by CPB2.Overexpression of WNT9A could reverse this injury and protect IPEC-J2 cells.Therefore,METTL3 could be used as a molecular target for the prevention and treatment of C.perfringens diarrhea in piglets.