Effect of PEDV infection on IPEC-J2 cells on miR-328-3p/FoxO4/NF-κB/MLCK signaling axis

Porcine epidemic diarrhea is a highly fatal and highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus. miRNA plays an important role in the pathogenesis of PEDV. Our previous study showed that miR-328-3p was up-regulated in PEDV infected Vero cells, and predicted that it could act on the NF-κB/MLCK signaling pathway by targeting and regulating FoxO4.Here, the targeting of miR-328-3p with FoxO4 was verified through dual-luciferase assay.We detected the mRNA expression of FoxO4, p65, MLCK, and MLC by qRT-PCR and the protein expression of FoxO4, p-FoxO4, p65, p-p65, IκBα, p-IκBα, MLCK, MLC, and p-MLC by Western-Blot after infection of the virus, respectively.The mRNA and protein expression of FoxO4 was detected by transfecting miR-328-3p and adding NF-κB pathway activator/inhibitor, and through this process, the expression of viral proteins and titers was detected. The results showed there was a targeting relationship between miR-328-3p and FoxO4. PEDV affects the transcription and expression of FoxO4, and activates key regulatory proteins in the NF-κB and MLCK/p-MLC signaling pathways, suggesting that PEDV can activate the NF-κB and MLCK/p-MLC signaling pathways in infected cells.miR-328-3p and NF-κB negatively regulated FoxO4,and they play a role in promoting viral replication.These data demonstrate that the miR-328-3p/FoxO4/NF-κB/MLCK signaling axis may play an important role in the pathogenesis of PEDV in IPEC-J2 cells.