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 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 miR-133a-3p. The figure shows the relative enrichment of miR-133a-3p in the ELAVL1-bound fraction compared to the IgG control. (C) Cell viability in different treatment groups was assessed using CCK8. Legend: The CCK8 assay was used to assess the cell viability of HUVEC and AC16 cells in different treatment groups. Cells were treated with N-EVs (50 µg/mL), miR-133a-3p inhibitor (100 nM), or a combination of both for 24 h. Cell viability was measured by assessing the absorbance at 450 nm. The figure shows the relative cell viability in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (D) Flow cytometry was employed to determine the apoptosis rate in different treatment groups. Legend: Flow cytometry was used to analyze the apoptosis rate in HUVEC and AC16 cells after treatment with N-EVs (50 µg/mL), miR-133a-3p inhibitor (100nM), or a combination of both for 24 h. Apoptosis was detected using Annexin V-FITC/PI double staining. The figure shows the proportions of early apoptotic (Annexin V + PI-), late apoptotic (Annexin V + PI+), and necrotic cells (Annexin V- PI+) in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (E, F) WB were utilized to examine the expression of pyroptosis and necroptosis markers in different treatment groups. Legend: (E) Western blot analysis was used to examine the expression of pyroptosis markers (cleaved caspase-1 and GSDMD) in HUVEC and AC16 cells after treatment with N-EVs (50 µg/mL), miR-133a-3p inhibitor (100 nM), or a combination of both for 24 h. The figure shows the protein expression levels of cleaved caspase-1 and GSDMD in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. Legend: (F) Western blot analysis was used to examine the expression of necroptosis markers (phosphorylated MLKL and RIPK3) in HUVEC and AC16 cells after treatment 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 phosphorylated MLKL and RIPK3 in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. Statistical significance was denoted as * vs. NC group (P < 0.05), # vs. inhibitor group (P < 0.05)