Fig. 7

ELAVL1 influences the formation of the PANoptosome by affecting the stability of NLRP3 mRNA. (A) The starBase database predicts the RBP protein of NLRP3. Legend: The starBase database was used to predict the RNA-binding proteins (RBPs) that interact with NLRP3 mRNA. The figure shows the predicted RBP proteins, with a focus on ELAVL1, which is known to play a role in mRNA stability and translation. The interaction scores and evidence from the database are provided to support the potential binding of ELAVL1 to NLRP3 mRNA. (B, C) QRT-PCR and WB were used to detect NLRP3 gene and protein expression (P < 0.05). Legend: (B) QRT-PCR was used to quantify the mRNA expression levels of NLRP3 in HUVEC and AC16 cells under different treatment conditions. Cells were treated with N-EVs (50 µg/mL), miR-133a-3p inhibitor (100 nM), or a combination of both for 24 h. The figure shows the relative mRNA expression levels of NLRP3, normalized to GAPDH. Legend: (C) Western blot analysis was used to examine the protein expression levels of NLRP3 in HUVEC and AC16 cells under different treatment conditions. Cells were treated with N-EVs (50 µg/mL), miR-133a-3p inhibitor (100nM), or a combination of both for 24 h. The figure shows the protein expression levels of NLRP3, with β-actin as the loading control. (D) RT-qPCR analysis of the decay rate of NLRP3 mRNA at the indicated times after ActD treatment. Legend: RT-qPCR was used to analyze the decay rate of NLRP3 mRNA in HUVEC and AC16 cells after treatment with actinomycin D (ActD, 5 µg/mL). Cells were treated with ActD for the indicated times (0, 2, 4, 6, and 8 h), and NLRP3 mRNA levels were quantified. The figure shows the relative mRNA levels of NLRP3 at each time point, normalized to GAPDH. (E) The ATtRACT database predicts ELAVL1 binding sites. Legend: The ATtRACT database was used to predict the binding sites of ELAVL1 on the NLRP3 mRNA. The figure shows the predicted binding sites within the 3’UTR of NLRP3 mRNA, highlighting the specific sequences and their positions. The binding scores and evidence from the database are provided to support the potential interaction between ELAVL1 and NLRP3 mRNA. (F, G) The targeting relationship between ELAVL1 and NLRP3 was confirmed by the dual-luciferase reporter assay. Legend: (F) The dual-luciferase reporter assay was used to confirm the direct targeting relationship between ELAVL1 and NLRP3. The 3’UTR of NLRP3 containing the putative ELAVL1 binding site was cloned into a luciferase reporter vector (NLRP3-WT). A mutant version of the 3’UTR with the binding site deleted (NLRP3-Mut) was also constructed. HEK293T cells were co-transfected with the reporter vectors and either ELAVL1 overexpression plasmid or a negative control (NC) plasmid. Luciferase activity was measured 48 h post-transfection. The figure shows the relative luciferase activity in the following groups: NC plasmid + NLRP3-WT, ELAVL1 overexpression plasmid + NLRP3-WT, NC plasmid + NLRP3-Mut, and ELAVL1 overexpression plasmid + NLRP3-Mut. Legend: (G) The dual-luciferase reporter assay was further used to confirm the targeting relationship between ELAVL1 and NLRP3 in the context of miR-133a-3p. HEK293T cells were co-transfected with the NLRP3-WT or NLRP3-Mut reporter vectors, ELAVL1 overexpression plasmid, and either miR-133a-3p mimic or a negative control (NC) mimic. Luciferase activity was measured 48 h post-transfection. The figure shows the relative luciferase activity in the following groups: NC mimic + NC plasmid + NLRP3-WT, miR-133a-3p mimic + NC plasmid + NLRP3-WT, NC mimic + ELAVL1 overexpression plasmid + NLRP3-WT, miR-133a-3p mimic + ELAVL1 overexpression plasmid + NLRP3-WT, NC mimic + NC plasmid + NLRP3-Mut, and miR-133a-3p mimic + NC plasmid + NLRP3-Mut. (H) RIP analysis of ELAVL1 protein binding to NLRP3 mRNA in cardiomyocytes. Legend: (H) RNA immunoprecipitation (RIP) assay was performed to validate the binding of ELAVL1 to NLRP3 mRNA in cardiomyocytes. Cardiomyocytes were lysed, and the cell lysates were incubated with magnetic beads conjugated with anti-ELAVL1 antibody or IgG (negative control). The enriched RNA was extracted and subjected to qRT-PCR to quantify the levels of NLRP3 mRNA. The figure shows the relative enrichment of NLRP3 mRNA in the ELAVL1-bound fraction compared to the IgG control. (I) Mechanism path diagram: Circulating extracellular vesicles regulate ELAVL1 by delivering miR-133a-3p, which affects NLRP3 mRNA stability, inhibiting PANoptosome formation. Legend: Schematic diagram illustrating the proposed mechanism by which circulating extracellular vesicles (EVs) regulate the expression and stability of NLRP3 mRNA through the delivery of miR-133a-3p. The diagram shows that N-EVs contain miR-133a-3p, which targets and reduces the expression of ELAVL1. Reduced ELAVL1 levels lead to decreased stability of NLRP3 mRNA, thereby inhibiting the formation of PANoptosomes and subsequent inflammatory responses