Circulating extracellular vesicles regulate ELAVL1 by delivering miR-133a-3p which affecting NLRP3 mRNA stability inhibiting PANoptosome formation

Figures

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  Fig. 1. Regulatory effects of EVs from different sources on cardiomyocytes and endothelial cells. (A) PKH67-labeled exosomes were used to investigate the cellular uptake of exosomes after 24 h of co-culture, and immunofluorescence was employed for detection. Legend: To investigate the cellular uptake of exosomes from different sources, PKH67 fluorescent dye was used to label the exosomes. After a 24-hour co-culture with HUVEC or AC16 cells, the intracellular fluorescence signals were observed using immunofluorescence microscopy to assess the efficiency of exosome uptake. The figure shows the uptake of exosomes from different sources, including the negative control group (no exosomes added) and the positive control group (PKH67-labeled exosomes added). Scale bar: 10 μm. (B) The cell proliferation ability of EVs derived from different sources on HUVEC and AC16 cells was assessed using CCK8 assay. Legend: The CCK8 assay was used to evaluate the impact of EVs from different sources on the proliferation of HUVEC and AC16 cells. The results are expressed as absorbance values at 450 nm, reflecting the degree of cell proliferation. (C) EdU assay was performed to evaluate the cell proliferation…

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  Fig. 2. Effects of EVs from different sources on cardiac function in a mouse model of acute myocardial infarction. (A) Schematic diagram of the in vivo experimental design. Legend: Schematic diagram of the in vivo experimental design. The timeline shows the key steps and time points for the myocardial infarction (MI) model, including surgery, treatment administration, and endpoint measurements. Mice were randomly divided into the control group and the treatment group. The treatment group received N-EVs (50 µg/mL) intravenously, while the control group received an equal volume of PBS. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured at 2 weeks and 4 weeks post-MI using echocardiography. The figure illustrates the overall experimental setup and timeline. (B-C) The left ventricular ejection fraction (LVEF) was assessed using echocardiography at baseline (1 day prior to myocardial infarction) and endpoint (3 weeks after myocardial infarction). Legend: Echocardiographic assessment of left ventricular ejection fraction (LVEF) was performed at baseline (1 day before myocardial infarction) and at the endpoint (3 weeks after myocardial…

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  Fig. 3. Improvement effect of N-EVs on AC16 and HUVEC after H2O2 treatment. (A) The cell proliferation ability of N-EVs on HUVEC and AC16 cells after H2O2 treatment was assessed using CCK8 assay. Legend: The CCK8 assay was used to evaluate the impact of normal extracellular vesicles (N-EVs) on the proliferation of HUVEC and AC16 cells after H2O2-induced oxidative stress. HUVEC and AC16 cells were pretreated with N-EVs (50 µg/mL) for 24 h, followed by exposure to H2O2 (200 µM) for another 24 h. Cell viability was assessed by measuring the absorbance at 450 nm. The figure shows the relative cell viability of HUVEC and AC16 cells in the following groups: control (no treatment), H2O2 treatment alone, and H2O2 treatment with N-EVs. (B) EdU assay was employed to evaluate the cell proliferation ability of EVs on HUVEC and AC16 cells after H2O2 treatment. Legend: The EdU (5-ethynyl-2’-deoxyuridine) assay was used to assess the impact of N-EVs on the proliferation of HUVEC and AC16 cells after H2O2-induced oxidative stress. HUVEC and AC16 cells were pretreated with N-EVs (50 µg/mL) for 24 h, followed by exposure to H2O2 (200 µM) for another 24 h. EdU incorporation into newly synthesized DNA was…

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  Fig. 4. The role of miR-133a-3p in EVs. (A) Volcano plots were employed to visualize differentially expressed miRNAs in the two types of circulating EVs. Legend: Volcano plots were generated to visualize the differentially expressed miRNAs in circulating EVs derived from normal (N-EVs) and acute myocardial infarction (AMI-EVs) samples. Each dot represents a specific miRNA, with the x-axis showing the log2 fold change (log2FC) and the y-axis showing the -log10 p-value. Red dots indicate significantly upregulated miRNAs, blue dots indicate significantly downregulated miRNAs, and gray dots represent non-significantly changed miRNAs. The figure highlights key miRNAs, including miR-133a-3p, miR-144-3p, and miR-23b-3p, which show differential expression between N-EVs and AMI-EVs. Data were normalized and analyzed using DESeq2. (B) The expression levels of miR-133a-3p, miR-144-3p, and miR-23b-3p in EVs derived from various sources were quantified using QRT-PCR. Legend: Quantitative real-time PCR (QRT-PCR) was used to quantify the expression levels of miR-133a-3p, miR-144-3p, and miR-23b-3p in EVs derived from different sources, including normal (N-EVs) and acute myocardial infarction (AMI-EVs)…

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  Fig. 5. The impact of miR-133a-3p on endothelial cells and cardiomyocytes. (A) The cell proliferation ability of N-EVs and miR-133a-3p inhibitor on HUVEC and AC16 was assessed using the CCK8 assay. Legend: The CCK8 assay was used to evaluate the impact of N-EVs and the miR-133a-3p inhibitor on the proliferation of HUVEC and AC16 cells. Cells were treated with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100nM) alone, or a combination of both for 24 h. Cell viability was assessed by measuring the absorbance at 450 nm. The figure shows the relative cell viability of HUVEC and AC16 cells in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (B) The cell proliferation ability of N-EVs and miR-133a-3p inhibitor on HUVEC and AC16 was evaluated by EdU staining. Legend: The EdU (5-ethynyl-2’-deoxyuridine) staining assay was used to assess the impact of N-EVs and the miR-133a-3p inhibitor on the proliferation of HUVEC and AC16 cells. Cells were treated with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100 nM) alone, or a combination of both for 24 h. EdU incorporation into newly synthesized DNA was detected using a…

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  Fig. 6. MiR-133a-3p regulates PANoptosis by targeting ELAVL1. (A) The targeting relationship between miR-133a-3p and ELAVL1 was confirmed by the dual-luciferase reporter assay. Legend: The dual-luciferase reporter assay was used to confirm the direct targeting relationship between miR-133a-3p and ELAVL1. The 3’ untranslated region (3’UTR) of ELAVL1 containing the putative miR-133a-3p binding site was cloned into a luciferase reporter vector (ELAVL1-WT). A mutant version of the 3’UTR with the binding site deleted (ELAVL1-Mut) was also constructed. HEK293T cells were co-transfected with the reporter vectors 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 + ELAVL1-WT, miR-133a-3p mimic + ELAVL1-WT, NC mimic + ELAVL1-Mut, and miR-133a-3p mimic + ELAVL1-Mut. (B) The mutual binding of miR-133a-3p and ELAVL1 was validated through the RIP assay. Legend: RNA immunoprecipitation (RIP) assay was performed to validate the mutual binding of miR-133a-3p and ELAVL1. HUVEC and AC16 cells were lysed, and the cell lysates were incubated with magnetic…

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  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…