Effects And Mechanism Of METTL3-mediated LncRNA EN₄2575 M⁶A Modification In The Injury Of IPEC-J2 Cells Induced By CPB2 Toxin

Piglet diarrhea is one of the common contagious diseases that lead to piglet mortality,severely affecting the healthy development of the pig industry and causing significant economic losses to the global pig farming industry.Clostridium perfringens type C is a common pathogenic bacterium that causes piglet diarrhea,and its CPB2toxin promotes the occurrence of diarrhea,resulting in necrotic enteritis in piglets.Currently,the prevention and treatment of Clostridium perfringens type C infection-induced diarrhea in piglets rely on vaccination and antibiotic therapy,which to some extent reduces the occurrence of piglet diarrhea.However,long-term use of vaccines and antibiotics can disrupt the intestinal microbiota of piglets,damage the intestinal barrier,and increase the antibiotic resistance of pathogenic bacteria.Therefore,improving the resistance of piglets to Clostridium perfringens type C infection by genetic nature is one of the effective ways to solve this type of piglet diarrhea.Lnc RNAs are involved in the regulation of bacterial infectious diseases in livestock and poultry,and m~6A modification plays an important role in pathogenic E.coli and viral infections causing diarrhea in livestock and poultry.However,whether m~6A-modified lnc RNAs are involved in diarrhea caused by Clostridium perfringens type C infection in piglets,and the regulatory role and molecular mechanism of their function are currently unknown.In view of this,this study used a cell model of Clostridium perfringens type C infection diarrhea in piglets constructed by our group previously,and examined the effect of CPB2 toxin treatment on RNA m~6A profiles of IPEC-J2 cells using Me RIP-seq technique.Then the m~6A-modified lnc RNAs associated with Clostridium perfringens infection resistance were screened by bioinformatics analysis.The regulatory roles and molecular mechanisms of key m~6A-modified lnc RNA EN＿42575(ENSSSCG00000042575)in CPB2 toxin-induced damage in IPEC-J2 cells were investigated using Me RIP-q PCR,FISH,Ed U,half-life,dual luciferase reporter assay,RNA pull down,and Western Blot.The main results of the study were as follows:1.RNA m~6A profiles of CPB2 toxin-treated IPEC-J2 cells were obtained using Me RIP-seq technique,and 6413 m~6A peaks were identified,corresponding to 5825m~6A-modified m RNAs and 433 m~6A-modified lnc RNAs.These m~6A peaks were mainly distributed in the 3’-UTR and stop codon regions,and the m~6A motif sequence GGACU was identified in both control and CPB2 groups.A total of 540 differentially expressed m~6A modified m RNAs(up regulated 155,down regulated 385)and 153differentially expressed m~6A modified lnc RNAs(up regulated 34,down regulated 119)were identified between the two groups.The differentially expressed m~6A-modified m RNAs were mainly enriched in Wnt signaling pathway,Hippo signaling pathway and other signaling pathways,and the differentially expressed m~6A-modified lnc RNA target genes were mainly involved in defense response to virus.The differentially expressed m~6A modified lnc RNA target genes are mainly involved in defense response to virus,and are significantly enriched in NF-kappa B signaling pathway,Cytokine-cytokine receptor interaction and Influenza A signaling pathway.The enrichment analysis of m~6A modified lnc RNA target genes by GO function and KEGG signaling pathway was used to screen lnc RNA EN＿42575,which is associated with viral defense response and immune response.2.The expression of lnc RNA EN＿42575 in IPEC-J2 cells significantly reduced(P<0.05)with increasing time of CPB2 toxin treatment and was lowest at 24 h.Nuclear/cytoplasmic isolation and FISH analysis indicated that lnc RNA EN＿42575 was mainly localized in the cytoplasm.CCK-8,Ed U,and LDH assays showed that overexpression of lnc RNA EN＿42575 promoted the proliferation of CPB2 toxin-treated IPEC-J2 cells and attenuated the cytotoxicity of IPEC-J2,while inhibition of lnc RNA EN＿42575 inhibited the proliferation of CPB2 toxin-treated IPEC-J2 cells and exacerbated the cytotoxicity of IPEC-J2.ΔΨm,ROS and RT-q PCR assay showed that overexpression of lnc RNA EN＿42575 inhibited CPB2-induced apoptosis of IPEC-J2cells and alleviated the oxidative damage of CPB2 toxin on IPEC-J2 cells,while interference with lnc RNA EN＿42575 promoted CPB2-induced apoptosis and oxidative damage in IPEC-J2 cells.3.Me RIP-q PCR results showed that CPB2 toxin treatment significantly reduced the m~6A level of lnc RNA EN＿42575 in IPEC-J2 cells(P<0.05),and IGV visualization analysis revealed that the two specific m~6A peaks that were highly enriched disappeared after CPB2 toxin treatment in IPEC-J2 cells.Overexpression of METTL3 significantly promoted the expression of lnc RNA EN＿42575,while inhibition of METTL3significantly decreased the expression of lnc RNA EN＿42575(P<0.05).The dual luciferase reporter gene assay indicated a targeting relationship between METTL3 and lnc RNA EN＿42575.The m~6A level of lnc RNA EN＿42575 was significantly reduced after interfering with METTL3.Half-life assay showed that inhibition of METTL3shortened the half-life of lnc RNA EN＿42575 and decreased the stability of lnc RNA EN＿42575.RNA pull down assay showed that lnc RNA EN＿42575 interacted with IFIT1 protein.Interference with lnc RNA EN＿42575 in CPB2 toxin-treated IPEC-J2cells suppressed IFIT1 protein expression.In summary,the m~6A profiles of CPB2 toxin-treated IPEC-J2 cells were obtained in this study,and the key m~6A-modified lnc RNA EN＿42575 involved in Clostridium perfringens type C infectious diarrhea in piglets was screened.The function of lnc RNA EN＿42575 in CPB2 toxin-induced IPEC-J2 cells was initially explored,and lnc RNA EN＿42575 was found to alleviate CPB2 toxin damage in IPEC-J2 cells.Mechanistic studies have shown that the methylesterase METTL3 promotes the expression of lnc RNA EN＿42575 in an m~6A-modified manner,which in turn affects IFIT1 protein expression in CPB2 toxin-induced IPEC-J2 cells.This study provides a reference molecular target for the prevention and control of Clostridium perfringens type C diarrhea in piglets,and provides some theoretical reference for solving Clostridium perfringens type C resistance breeding in piglets.