FTO Regulates Apoptosis in CPB2-Treated IPEC-J2 Cells by Targeting Caspase 3 Apoptotic Protein

Simple Summary Fat mass and obesity associated protein (FTO) is a key demethylase in the process of bacterial diarrhea in piglets. However, the involvement of FTO in infectious diarrhea caused by Clostridium perfringens type C is not known. This study demonstrated that FTO plays an indispensable role in this type of diarrhea; and that the absence of FTO promotes apoptosis and inflammation in the intestinal porcine epithelial cell line-J2 (IPEC-J2). FTO targets Caspase 3 to affect the apoptosis of IPEC-J2 cells. 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.

Automated summary

This study demonstrated the critical role of FTO in infectious diarrhea caused by Clostridium perfringens type C, showing that FTO deficiency promotes apoptosis and inflammation. FTO was found to target Caspase 3, affecting the apoptosis of IPEC-J2 cells. Moreover, m6A modification of mRNA was shown to play a significant role in this process.