Regulatory Effect of Methylation of the Porcine AQP3 Gene Promoter Region on Its Expression Level and Porcine Epidemic Diarrhea Virus Resistance

Abstract

As an important carrier for intestinal secretion and water absorption, aquaporin 3 (AQP3) is closely related to diarrhea. In this study, we investigated the mechanisms of AQP3 gene expression regulation in porcine epidemic diarrhea virus (PEDV)-induced diarrhea confirmed by PCR amplification and sequencing. Evaluation of intestinal pathology showed that diarrhea caused by PEDV infection destroyed the intestinal barrier of piglets. qPCR analysis showed that AQP3 expression in the small intestine of PEDV-infected piglets was extremely significantly decreased. qPCR and Bisulfite sequencing PCR revealed an increase in the methylation levels of both CpG islands in the AQP3 promoter region in the jejunum of PEDV-infected piglets. The methylation of mC-20 and mC-10 sites within the two CpG islands showed a significant negative correlation with AQP3 expression. Chromatin Co-Immunoprecipitation (ChIP)-PCR showed that the Sp1 transcription factor was bound to the AQP3 promoter region containing these two CpG sites. AQP3 expression was also extremely significantly reduced in Sp1-inhibited IPEC-J2 cells, indicating that abnormal methylation at the mC-20 site of CpG1 and the mC-10 site of CpG2 reduces its expression in PEDV-infected piglet jejunum by inhibiting the binding of Sp1 to the AQP3 promoter. These findings provide a theoretical basis for further functional studies of porcine AQP3.