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) samples. The figure shows the relative expression levels of these miRNAs, normalized to U6 snRNA. (C) The abundance of miR-133a-3p was assessed after treatment with AMI-EV or N-EV at a concentration of 5 µg/mL. Legend: The abundance of miR-133a-3p was measured in HUVEC and AC16 cells after treatment with AMI-EVs or N-EVs at a concentration of 5 µg/mL for 24 h. QRT-PCR was used to quantify the miR-133a-3p levels, normalized to U6 snRNA. The figure shows the relative expression levels of miR-133a-3p in the control (no EV treatment), N-EV treatment, and AMI-EV treatment groups. (D) The level of miR-133a-3p expression was determined in RNase A-treated EVs (0.1 mg/mL). Legend: The stability of miR-133a-3p in EVs was assessed by treating EVs with RNase A (0.1 mg/mL) for 30 min at 37 °C. QRT-PCR was used to quantify the miR-133a-3p levels in RNase A-treated and untreated EVs, normalized to U6 snRNA. The figure shows the relative expression levels of miR-133a-3p in the control (untreated EVs) and RNase A-treated EVs. (E) QRT-PCR analysis was performed to evaluate the presence of miR-133a-3p in Triton X-100-treated (0.3%) and RNase-digested EVs. Legend: The integrity and protection of miR-133a-3p within EVs were evaluated by treating EVs with Triton X-100 (0.3%) and RNase A (0.1 mg/mL). QRT-PCR was used to quantify the miR-133a-3p levels in the following groups: control (untreated EVs), Triton X-100-treated EVs, and RNase A-digested EVs, normalized to U6 snRNA. The figure shows the relative expression levels of miR-133a-3p in each group. (F) The impact of EV treatment dose on the expression level of miR-133a-3p in AC16 and HUVEC cells was investigated through time-dependent and dose-replacement experiments. Legend: Time-dependent and dose-replacement experiments were conducted to investigate the impact of EV treatment on the expression level of miR-133a-3p in AC16 and HUVEC cells. HUVEC and AC16 cells were treated with N-EVs or AMI-EVs at different concentrations (1, 5, and 10 µg/mL) for various time points (24, 48, and 72 h). QRT-PCR was used to quantify the miR-133a-3p levels, normalized to U6 snRNA. The figure shows the relative expression levels of miR-133a-3p in the following groups: control (no EV treatment), N-EV treatment, and AMI-EV treatment at different doses and time points. Statistical significance was determined by comparing with AMI-EVs group (*P < 0.05)