仔猪腹泻是一种猪疾病,对养猪业造成严重的经济影响。产气荚膜梭菌β2毒素(CPB2),这是C型产气荚膜梭菌的主要毒素,可能会导致许多家畜肠道疾病。N6-甲基腺苷(m6A)RNA甲基化在家畜和其他动物的许多免疫和炎性疾病中起关键作用。然而,尚未研究m6A甲基化在暴露于CPB2的猪肠上皮细胞(IPEC-J2)中的作用。为了解决这个问题,我们用CPB2毒素处理IPEC-J2细胞,然后通过RT-qPCR定量甲基化相关酶的表达,并通过比色N6-甲基腺苷定量评估样品的m6A甲基化状态.结果表明,甲基化酶有不同程度的变化,而m6A甲基化水平增加(p<0.01)。在此基础上,我们进行了N6-甲基腺苷测序(m6A-seq)和RNA测序(RNA-seq),以检测CPB2毒素暴露后IPEC-J2细胞的详细m6A修饰和基因表达.我们的结果表明,1,448个m6A修饰位点,包括437个上调和1011个下调,CPB2毒素暴露细胞和未暴露细胞之间存在显着差异(p<0.05)。KEGG通路分析结果显示,m6A峰上调基因(n=394)主要富集于肿瘤,库欣综合征与Wnt信号通路,而m6A峰下调基因(n=920)主要与细胞凋亡有关,小细胞肺癌,和单纯疱疹病毒1型感染信号通路。此外,基因表达(RNA-seq数据)分析确定了1,636个差异表达基因(DEG),其中与对照组相比,毒素暴露组1,094例上调,542例下调。此外,下调的基因参与了Hippo和Wnt信号通路。有趣的是,m6A-seq和RNA-seq的联合结果鉴定了m6A峰上调但表达下调的基因,这里称为“超下降”基因(n=18),主要富集在Wnt信号通路中。因此,我们推测,在CPB2诱导的IPEC-J2细胞中,Wnt信号通路中的基因可能被m6A甲基化修饰。这些发现提供了新的见解,能够进一步探索CPB2毒素引起的仔猪腹泻的潜在机制。
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. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A 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 m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A 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 m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A 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 m6A-seq and RNA-seq identified genes with up-regulated m6A 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 m6A 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.