Yang Jiaojiao, Zhang Juanli, Gao Xiaoli, Luo Ruirui, Xie Kaihui, Wang Wei, Li Jie, Yang Qiaoli, Huang Xiaoyu, Yan Zunqiang, Wang Pengfei, Gun Shuangbao
College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China.
College of Animal Science and Technology, Northwest A&F University, Xi'an 712100, China.
Animals (Basel). 2022 Jun 26;12(13):1644. doi: 10.3390/ani12131644.
N6-methyladenosine (m6A) modification can accommodate mRNA processing, stability, and translation in mammals, and fat mass and obesity associated protein (FTO) is a vital demethylase in the m6A modification pathway. Clostridium perfringens type C (C. perfringens type C) causes diarrhea in piglets and has a serious impact on the pig industry. However, our understanding of the effect of m6A in the process of C. perfringens type C infectious piglet diarrhea (CPTCIPD) is limited. Here, an in vitro model of CPTCIPD was constructed by treating the intestinal porcine epithelial cell line-J2 (IPEC-J2) with Clostridium perfringens beta2 (CPB2) toxin, and the role of FTO was analyzed using quantitative real-time polymerase chain reaction, Western blotting, and flow cytometry. The results revealed that the overall RNA m6A contents at the tissue and cell levels were significantly up-regulated after C. perfringens infection (p < 0.05). FTO expression was significantly reduced in CPB2-treated IPEC-J2 cells. Functionally, FTO knockdown in the treated cells inhibited their proliferation and promoted apoptosis and the inflammation phenotype, whereas FTO overexpression had the opposite effects. Inhibiting FTO prolonged the half-life and up-regulated the expression of Caspase 3, leading to apoptosis. Therefore, this work explored the regulation of FTO in IPEC-J2 cells after CPB2 treatment and enhanced our understanding of the effect of the m6A modification in CPTCIPD.
N6-甲基腺苷（m6A）修饰可调控哺乳动物体内mRNA的加工、稳定性及翻译过程，而脂肪量和肥胖相关蛋白（FTO）是m6A修饰途径中的一种重要去甲基化酶。C型产气荚膜梭菌可导致仔猪腹泻，对养猪业造成严重影响。然而，我们对m6A在C型产气荚膜梭菌感染仔猪腹泻（CPTCIPD）过程中的作用了解有限。在此，通过用C型产气荚膜梭菌β2（CPB2）毒素处理猪小肠上皮细胞系-J2（IPEC-J2）构建了CPTCIPD的体外模型，并运用定量实时聚合酶链反应、蛋白质免疫印迹法及流式细胞术分析了FTO的作用。结果显示，产气荚膜梭菌感染后，组织和细胞水平的总体RNA m6A含量显著上调（p < 0.05）。在CPB2处理的IPEC-J2细胞中，FTO表达显著降低。在功能上，处理后的细胞中FTO基因敲低抑制了细胞增殖，促进了细胞凋亡及炎症表型，而FTO过表达则产生相反的效果。抑制FTO可延长半胱天冬酶3的半衰期并上调其表达，从而导致细胞凋亡。因此，本研究探索了CPB2处理后FTO在IPEC-J2细胞中的调控作用，增进了我们对m6A修饰在CPTCIPD中作用的理解。
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