MiR-129a-3p Inhibits PEDV Replication by Targeting the EDA-Mediated NF-κB Pathway in IPEC-J2 Cells.

Shenglong Wu,Zhengchang Wu,Xiaoyi Qi,Yue Cao,Wenbin Bao

doi:10.3390/ijms22158133

Abstract

Previous studies have shown that microRNAs (miRNAs) are closely related to many viral infections. However, the molecular mechanism of how miRNAs regulate porcine epidemic diarrhea virus (PEDV) infection remains unclear. In this study, we first constructed a PEDV-infected IPEC-J2 cytopathic model to validate the relationship between miR-129a-3p expression levels and PEDV resistance. Secondly, we explored the effect of miR-129a-3p on PEDV infection by targeting the 3′UTR region of the ligand ectodysplasin (EDA) gene. Finally, transcriptome sequencing was used to analyze the downstream regulatory mechanism of EDA. The results showed that after 48 h of PEDV infection, IPEC-J2 cells showed obvious pathological changes, and miR-129a-3p expression was significantly downregulated (p < 0.01). Overexpression of miR-129a-3p mimics inhibited PEDV replication in IPEC-J2 cells; silencing endogenous miR-129a-3p can promote viral replication. A dual luciferase assay showed that miR-129a-3p could bind to the 3′UTR region of the EDA gene, which significantly reduced the expression level of EDA (p < 0.01). Functional verification showed that upregulation of EDA gene expression significantly promoted PEDV replication in IPEC-J2 cells. Overexpression of miR-129a-3p can activate the caspase activation and recruitment domain 11 (CARD11) mediated NF-κB pathway, thus inhibiting PEDV replication. The above results suggest that miR-129a-3p inhibits PEDV replication in IPEC-J2 cells by activating the NF-κB pathway by binding to the EDA 3′UTR region. Our results have laid the foundation for in-depth study of the mechanism of miR-129a-3p resistance and its application in porcine epidemic diarrhea disease-resistance breeding.

Highlights

Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV) [1,2]
TCID50 experiments showed that PEDV infected J2 cells had significantly fewer infected virions after transfection with miR-129a-3p mimics (p < 0.01), virions infected with miR-129a-3p inhibitor were efficiently increased (p < 0.01) (Figure 1E)
Western blot was used to detect the expression of PEDV N protein (QNL15265.1), and it was found that the expression of PEDV N protein was significantly lower in the mimics group compared with that of the NC group (Figure 1G); Int

Summary

Introduction

Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV) [1,2]. Studies have found that miRNAs play an important role in many biological processes, such as cell proliferation, differentiation, apoptosis, stress response, and inflammatory pathway regulation [8,9,10,11,12,13]. MiRNA expression in host cells is significantly affected during virus infection. The host cell changes miRNA expression, which may be due to the virus escaping the antiviral immune response by changing the intracellular environment. Recognition of the miRNA-mediated regulation mechanism of PEDV infection will help to identify new targets for anti-PEDV treatment. The aim was to reveal a new molecular mechanism by which miR-129a-3p regulates PEDV resistance and lay the foundation for in-depth study of the application of miR-129a-3p in breeding for disease resistance to porcine epidemic diarrhea

Effect of miR-129a-3p on PEDV Viral Replication

Effect of the EDA Gene on PEDV Viral Replication

Mechanism of Downstream Regulation of the EDA Gene

Discussion

Plasmid Transfection

Dual Luciferase-Reporter Assays

TCID50 Analysis

EDA Overexpression Cell Lines

RT-qPCR Analysis

Western Blot Analysis

RNA-seq and Computational Analysis