The neuropathological mechanism of EV-A71 infection attributes to inflammatory pryoptosis and viral replication via activating the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis

Figures

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  Fig. 1. EV-A71 infection triggers cell death of SH-SY5Y cells. (A) The percentage of cells undergoing cell death was calculated through Annexin V-FITC/PI double staining. (B) The cell viability was conducted with Cell Counting Kit-8. (C) Levels of LDH released into the cell culture medium from membrane pores were examined using an LDH Cytotoxicity Assay Kit. (D) Caspase1 activity was assessed with a Caspase1 Activity Assay Kit. .

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  Fig. 2. EV-A71 infection induces NLRP3-dependent pyroptosis in SH-SY5Y cells. (A) NLRP3, ASC, Casepase1, Gasdermin D, IL-1β and IL-18 mRNA levels were analyzed by qRT-PCR. (B) The abundance of NLRP3, ASC, pro-Caspase1, cleaved Caspase 1, Gasdermin D, IL-1β and IL-18 was determined by WB analysis. (C) At 48 and 72 h post infection, infected SH-SY5Y cells were subjected to indirect immunofluorescence assays with anti-VP1 (red) and anti-Caspase-1 (green) antibodies. (D) Secretion of IL-1β and IL-18 upon infection with EV-A71 was measured with a commercial ELISA kit. .

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  Fig. 3. EV-A71 infection activates the release of inflammatory cytokines. Flow cytometry results for 12 cytokines during EV-A71 infection. .

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  Fig. 4. Identification of differentially expressed circRNAs. (A) The number and genomic origin of differentially expressed circRNAs at 24 hpi with EV-A71 infection. (B) The number and genomic origin of differentially expressed circRNAs at 48 hpi with EV-A71 infection.

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  Fig. 5. Specifical expression profiles of circRNAs induced by EV-A71 infection in SH-SY5Y cells. (A) Venn diagram displaying the common and differential circRNAs during EV-A71 infection at 24 hpi and 48 hpi. (B) Hierarchical cluster analysis of differentially expressed circRNAs between control and EV-A71-infected groups. (C) Trend analysis of 211 overlapping differentially expressed circRNAs.

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  Fig. 6. Functional analysis of the persistently up-regulated circRNAs. (A) GO enrichment analysis for the parent genes of the persistently up-regulated circRNAs. (B) KEGG enrichment analysis for the parent genes of the persistently up-regulated circRNAs. (C) Construction of circRNA-medicated networks associated with NLRPs. (D) Bioinformatic analysis was performed to predict the interaction between pathogens and pyroptosis-related inflammatory pathway. .

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  Fig. 7. has_circ_0045431/has-miR-584/NLRP3 regulatory axis involves in EV-A71-induced pyroptotic cell death. (A) The cell viability of SH-SY5Y cells was assessed by CCK8 kit. (B) The cell damage of SH-SY5Y cells was evaluated by LDH cytotoxicity assay kit. (C) The Caspase 1 activity of SH-SY5Y cells was examined by Caspase-1 activity assay kit. .

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  Fig. 8. has_circ_0045431/has-miR-584/NLRP3 regulatory axis promotes EV-A71-induced pyroptosis formation. (A) SH-SY5Y cells with different treatments were subjected to WB analysis for NLRP3, ASC, pro-Caspase1, cleaved Caspase 1, Gasdermin D, IL-1β and IL-18 expression. (B) The cell culture supernatant was subjected to the examination of ELISA for IL-1β and IL-18 concentration.

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  Fig. 9. has_circ_0045431/has-miR-584/NLRP3 regulatory axis enhances inflammatory response during EV-A71 infection. Flow cytometry analysis of TNF-α, IL-12, IL-4, IL-17, IL-8, IFN-γ, IL-10, IL-1β, IL-6, IL-2, IFN-α and IL-5 in SH-SY5Y cells with different plasmids transfection. .

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  Fig. 10. The effect of has_circ_0045431/has-miR-584/NLRP3 regulatory axis on EV-A71 replication. (A) The viral genome copies of EV-A71 were determined by Taqman based qRT-PCR. (B) The virus titers of EV-A71 were evaluated with a plaque assay. (C) Cell lysates were prepared for WB to measure VP1 protein in treated SH-SY5Y cells.