Zhu Kaiqing, Liu Puyu, Liu Kangping, Cui Yanan, Jiang Pengxin, Wang Xutao, Chen Ning, Cui Jiamei, Hou Zijuan, Li Jianguo, Fan Jinghui, Zuo Yuzhu, Li Yan
College of Veterinary Medicine, Hebei Agricultural University, Baoding, China.
College of Animal Science and Technology, Hebei Agricultural University, Baoding, China.
Front Vet Sci. 2025 Jun 4;12:1578941. doi: 10.3389/fvets.2025.1578941. eCollection 2025.
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.
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.
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.
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.
产肠毒素大肠杆菌（ETEC）是引起仔猪腹泻的主要病原体。本研究旨在探讨猪环状RNA（circRNA）在ETEC感染期间调节肠道免疫的机制。
通过RNA测序获得ETEC感染和未感染的IPEC-J2细胞中的circRNA表达谱。使用qRT-PCR和RNase R消化方法验证circRNA的稳定共价闭合结构。使用Miranda软件、双荧光素酶报告基因检测、靶基因或RNA的敲低和过表达分析潜在的circRNA/miRNA/mRNA相互作用。通过qRT-PCR和蛋白质印迹评估焦亡相关因子的表达。利用流式细胞术定量焦亡细胞，并使用透射电子显微镜观察细胞形态。
在本研究中，在ETEC感染的与未感染的IPEC-J2细胞中总共鉴定出328个差异表达的circRNA，其中一个名为circ_0020647的新型circRNA在感染后显著上调。circ_0020647由基因间序列编码，形成共价闭合环结构。我们证明circ_0020647通过直接结合作为miRNA ssc-mir-185的分子海绵，进而靶向BRD4 mRNA。ETEC感染后，circ_0020647通过增加NLRP3、GSDMD和caspase-1的表达促进IPEC-J2细胞中的焦亡。此外，circ_0020647参与ETEC诱导的细胞损伤，其特征在于乳酸脱氢酶外流、IL-1β和IL-18分泌、膜孔形成和线粒体异常。我们揭示了circ_0020647在调节焦亡中的作用是由ssc-mir-185/BRD4轴介导的。
我们的研究构建了一个新型的circ_0020647/ssc-mir-185/BRD4网络，该网络在ETEC感染诱导的IPEC-J2细胞焦亡中起重要作用。我们的研究结果表明，circRNA/miRNA/mRNA网络可能是ETEC引起的仔猪腹泻的新型生物标志物和潜在治疗靶点。
