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Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry.  Clostridium perfringens  beta2 toxin (CPB2), which is a major toxin of  C. perfringens  type C, may cause intestinal diseases in many domestic animals. N 6 -methyladenosine (m 6 A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m 6 A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m 6 A methylation status of the samples by colorimetric N 6 -methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m 6 A methylation level increased ( p  < 0.01). On this basis, we performed N 6 -methyladenosine sequencing (m 6 A-seq) and RNA sequencing (RNA-seq) to examine the detailed m 6 A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m 6 A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells ( p  < 0.05). KEGG pathway analysis results showed that m 6 A peaks up-regulated genes ( n  = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m 6 A peaks down-regulated genes ( n  = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m 6 A-seq and RNA-seq identified genes with up-regulated m 6 A peaks but with down-regulated expression, here referred to as “hyper-down” genes ( n  = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m 6 A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

frontiers in genetics

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing