A novel circular RNA circ_0020647 promotes ETEC-induced IPEC-J2 cell pyroptosis via the ssc-miR-185/BRD4 axis.

Abstract

INTRODUCTION: Enterotoxigenic(ETEC) is a major pathogen causing piglet diarrhea. This study aimed to investigate the mechanism of porcine circular RNAs (circRNAs) in regulating intestinal immunity during ETEC infection. METHODS: The circRNA expression profiles were obtained in ETEC-infected and uninfected IPEC-J2 cells via RNA-sequencing. The stable covalently closed structure of circRNAs was validated using qRT-PCR and RNase R digestion methods. The potential circRNA/miRNA/mRNA interactions were analyzed using Miranda software, dual-luciferase reporter assay, knockdown and over-expression of the target gene or RNA. The expression of pyroptosis-related factors was assessed by qRT-PCR and Western blot. Flow cytometry was utilized to quantify pyroptotic cells, and transmission electron microscopy was used to observe cellular morphology. RESULTS: In this study, a total of 328 differentially expressed circRNAs were identified in ETEC-infected versus uninfected IPEC-J2 cells, among which a novel circRNA named circ_0020647 was significantly upregulated post-infection. Circ_0020647, encoded by an intergenic sequence, forms a covalently closed loop structure. We demonstrated that circ_0020647 acts as a molecular sponge for miRNA ssc-mir-185 through direct binding, which in turn targets BRD4 mRNA. Following ETEC infection, circ_0020647 promoted pyroptosis in IPEC-J2 cells by increasing the expression of NLRP3, GSDMD, and caspase-1. Additionally, circ_0020647 was involved in ETEC-induced cell injury, characterized by LDH efflux, IL-1β and IL-18 secretion, formation of membrane pores, and mitochondrial abnormalities. We revealed that the role of circ_0020647 in regulating pyroptosis was mediated by the ssc-mir-185/BRD4 axis. CONCLUSION: Our study constructed a novel circ_0020647/ssc-mir-185/BRD4 network that played an important role in the pyroptosis of IPEC-J2 cells induced by ETEC infection. Our findings imply that the circRNA/miRNA/mRNA network may be a novel biomarker and a potential therapeutic target for diarrhea in piglets caused by ETEC.