The Competitive Endogenous RNA (ceRNA) Regulation in Porcine Alveolar Macrophages (3D4/21) Infected by Swine Influenza Virus (H1N1 and H3N2).

H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.

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

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.

miR-215 Targeting Novel Genes EREG, NIPAL1 and PTPRU Regulates the Resistance to E.coli F18 in Piglets.

Previous research has revealed that miR-215 might be an important miRNA regulating weaned piglets' resistance to Escherichia coli (E. coli) F18. In this study, target genes of miR-215 were identified by RNA-seq, bioinformatics analysis and dual luciferase detection. The relationship between target genes and E. coli infection was explored by RNAi technology, combined with E. coli stimulation and enzyme linked immunosorbent assay (ELISA) detection. Molecular regulating mechanisms of target genes expression were analyzed by methylation detection of promoter regions and dual luciferase activity assay of single nucleotide polymorphisms (SNPs) in core promoter regions. The results showed that miR-215 could target EREG, NIPAL1 and PTPRU genes. Expression levels of three genes in porcine intestinal epithelial cells (IPEC-J2) in the RNAi group were significantly lower than those in the negative control pGMLV vector (pGMLV-NC) group after E. coli F18 stimulation, while cytokines levels of TNF-α and IL-1ß in the RNAi group were significantly higher than in the pGMLV-NC group. Variant sites in the promoter region of three genes could affect their promoter activities. These results suggested that miR-215 could regulate weaned piglets' resistance to E. coli F18 by targeting EREG, NIPAL1 and PTPRU genes. This study is the first to annotate new biological functions of EREG, NIPAL1 and PTPRU genes in pigs, and provides a new experimental basis and reference for the research of piglets disease-resistance breeding.