Department of Respiratory Medicine, The First People's Hospital of Yunnan Province, Kunming, PR China; The Affiliated Hospital of Kunming University of Science and Technology, Kunming, PR China.
Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, PR China.
Virus Res. 2023 Oct 2;335:199195. doi: 10.1016/j.virusres.2023.199195. Epub 2023 Aug 15.
Neuropathological damage has been considered to be the main cause of death from EV-A71 infection, but the underlying mechanism has not been elucidated. Pyroptosis, a new form of inflammatory programmed cell death, has been verified to be involved in the pathogenesis of various viruses. circRNAs are a novel type of endogenous noncoding RNA gaining research interest in recent years, especially their special roles in the process of virus infection. Thus, in this study, we combined EV-A71, pyroptosis and circRNA to find a breakthrough in the pathogenesis of EV-A71 infection. Firstly, whether EV-A71 infection leaded to pyroptosis formation was examined by a series detection of cell death, cell viability, LDH release, caspase 1 activity, the expression levels of pyroptosis-related molecules and the concentrations of IL-1β and IL-18. Secondly, high-throughput sequencing of circRNAs was carried out to excavate the circRNA-miRNA-mRNA regulatory axis which might be associated with pyroptosis formation. Finally, the gain- and loss-of-functional experiments were further conducted to identify their functions. Our results showed that EV-A71 infection caused pyroptosis formation in SH-SY5Y cells. The circRNA sequencing analyzed the differentially expressed circRNAs and their possible functions. It was found that the hsa_circ_0045431/hsa_miR_584/NLRP3 regulatory axis might be involved in pyroptosis formation during EV-A71 infection. Then, hsa_circ_0045431 sponged hsa_miR_584 and hsa_miR_584 directly targeted NLRP3 were validated by IF, dual-luciferase, qRT-PCR and WB assays. Functional experiments were performed to further uncover that the up-regulation of hsa_circ_0045431 and NLRP3 promoted the inflammatory pyroptosis and viral replication, while the up-regulation of hsa_miR_584 suppressed the inflammatory pyroptosis and viral replication, and vice versa. Collectively, our study demystified that EV-A71 infection induced pyroptosis formation by activating hsa_circ_0045431/hsa_miR_584/NLRP3 regulatory axis, which could further effect viral replication. These findings provided novel insights into the pathogenesis of EV-A71 infection, and meanwhile revealed that the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis can serve as a potential biological therapeutic target for EV-A71 infection.
神经病理学损伤被认为是 EV-A71 感染导致死亡的主要原因，但潜在机制尚未阐明。细胞焦亡是一种新的炎症程序性细胞死亡形式，已被证实参与了各种病毒的发病机制。circRNAs 是近年来受到关注的一种新型内源性非编码 RNA，特别是它们在病毒感染过程中的特殊作用。因此，在这项研究中，我们将 EV-A71、细胞焦亡和 circRNA 结合起来，以期在 EV-A71 感染的发病机制方面取得突破。首先，通过一系列细胞死亡、细胞活力、LDH 释放、半胱天冬酶 1 活性、细胞焦亡相关分子的表达水平以及白细胞介素-1β和白细胞介素-18 的浓度检测，研究 EV-A71 感染是否导致细胞焦亡的形成。其次，通过高通量测序对 circRNAs 进行分析，挖掘与细胞焦亡形成相关的 circRNA-miRNA-mRNA 调控轴。最后，通过功能获得和功能丧失实验进一步确定它们的功能。我们的研究结果表明，EV-A71 感染导致 SH-SY5Y 细胞发生细胞焦亡。circRNA 测序分析了差异表达的 circRNAs 及其可能的功能。结果发现，hsa_circ_0045431/hsa_miR_584/NLRP3 调控轴可能参与了 EV-A71 感染期间的细胞焦亡形成。然后，通过 IF、双荧光素酶报告基因、qRT-PCR 和 WB 检测验证了 hsa_circ_0045431 可以海绵吸附 hsa_miR_584，并且 hsa_miR_584 可以直接靶向 NLRP3。进一步进行功能实验以揭示 hsa_circ_0045431 和 NLRP3 的上调促进了炎症性细胞焦亡和病毒复制，而 hsa_miR_584 的上调抑制了炎症性细胞焦亡和病毒复制，反之亦然。综上所述，本研究揭示了 EV-A71 感染通过激活 hsa_circ_0045431/hsa_miR_584/NLRP3 调控轴诱导细胞焦亡形成，从而进一步影响病毒复制。这些发现为 EV-A71 感染的发病机制提供了新的见解，同时也表明 hsa_circ_0045431/hsa_miR_584/NLRP3 调控轴可以作为 EV-A71 感染的潜在生物治疗靶点。
