Journal: iScience
Article Title: Adipose extracellular vesicles carrying miR-210-3p drive macrophage inflammation and nicotine-induced atherosclerosis
doi: 10.1016/j.isci.2026.115151
Figure Lengend Snippet: miR-210-3p promotes macrophage inflammatory activation and foam cell formation through KLF7 suppression, which is reversed by KLF7 overexpression (A) Venn diagram showing 10 common downstream target genes of miR-210-3p identified from three databases (TarBase, mirDIP, and TargetScan); KLF7 was selected as a key target for further investigation. Potential binding sites of miR-210-3p on the KLF7 mRNA 3′ untranslated region (3′UTR) (3′UTR) are also shown. (B) Dual-luciferase reporter assay in HEK293T cells co-transfected with miR-210-3p mimics (or NC mimics) and luciferase vectors containing wild-type (Klf7-WT) or Mutant (Klf7-MT) 3′UTR ( n = 9). (C–E) Validation of KLF7 suppression in macrophages. Western blot images (C), quantification of KLF7/GAPDH protein ratio (D, n = 6), and qRT-PCR analysis of Klf7 mRNA (E, n = 3) after transfection with miR-210-3p mimics or inhibitors. (F and G) Representative immunofluorescence images (F) and quantification (G) of KLF7 expression after transfection with miR-210-3p mimics or inhibitors (KLF7, red; nuclei, blue [DAPI]; n = 6; scale bars, 20 μm). (H and I) RT-qPCR (H, n = 6) and western blot (I, n = 3) analyses confirming transduction efficiency and KLF7 overexpression in macrophages following lentiviral transduction with Lv-KLF7 or empty lentivirus (control). (J and K) qRT-PCR analysis of IL-1β and IL-6 mRNA expression in macrophages post-transfection with miR-210-3p mimics and/or transduction with Lv-KLF7 ( n = 6). (L and M) Representative immunofluorescence images (L) and quantification (M) of KLF7 expression in RAW264.7 macrophages transfected with miR-210-3p mimics and transduced with Lv-KLF7 (KLF7, green; nuclei, blue [DAPI]; n = 4; scale bars, 50 μm). (N and O) Representative DCFH-DA fluorescence images (N) and quantification (O) of intracellular ROS levels (green). Red fluorescence (mCherry) indicates macrophages successfully transduced with the lentiviral vectors. Note that KLF7 overexpression attenuates miR-210-3p-induced oxidative stress. (scale bars, 50 μm; n = 6). (P and Q) Representative BODIPY-cholesterol fluorescence images (P) and quantification of intracellular fluorescence intensity (Q) in macrophages transfected with miR-210-3p mimics and transduced with Lv-KLF7 ( n = 3; scale bars, 50 μm). (R and S) Representative oil red O staining (R) and semi-quantitative analysis of oil red O-positive lipid area (S) in macrophages transfected with miR-210-3p mimics and transduced with Lv-KLF7 ( n = 5; scale bars, 100 μm). Values are shown as mean ± SEM. Two-group comparisons were performed using unpaired, two-tailed Student’s t tests, and multi-group comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple-comparisons post-hoc test. Sample sizes (n) indicate biological replicates per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Article Snippet: Lentiviral negative control virus , GeneChem , Vector: GV737; CMV-MCS-EF1a-mCherry-T2A-puromycin.
Techniques: Activation Assay, Over Expression, Binding Assay, Luciferase, Reporter Assay, Transfection, Mutagenesis, Biomarker Discovery, Western Blot, Quantitative RT-PCR, Immunofluorescence, Expressing, Transduction, Control, Fluorescence, Staining, Two Tailed Test
![miR-210-3p promotes macrophage inflammatory activation and foam cell formation through KLF7 suppression, which is reversed by KLF7 overexpression (A) Venn diagram showing 10 common downstream target genes of miR-210-3p identified from three databases (TarBase, mirDIP, and TargetScan); KLF7 was selected as a key target for further investigation. Potential binding sites of miR-210-3p on the KLF7 mRNA 3′ untranslated region (3′UTR) (3′UTR) are also shown. (B) Dual-luciferase reporter assay in HEK293T cells co-transfected with miR-210-3p mimics (or NC mimics) and luciferase vectors containing wild-type (Klf7-WT) or Mutant (Klf7-MT) 3′UTR ( n = 9). (C–E) Validation of KLF7 suppression in macrophages. Western blot images (C), quantification of KLF7/GAPDH protein ratio (D, n = 6), and qRT-PCR analysis of Klf7 mRNA (E, n = 3) after transfection with miR-210-3p mimics or inhibitors. (F and G) Representative immunofluorescence images (F) and quantification (G) of KLF7 expression after transfection with miR-210-3p mimics or inhibitors (KLF7, red; nuclei, blue [DAPI]; n = 6; scale bars, 20 μm). (H and I) RT-qPCR (H, n = 6) and western blot (I, n = 3) analyses confirming transduction efficiency and KLF7 overexpression in macrophages following <t>lentiviral</t> transduction with Lv-KLF7 or empty lentivirus (control). (J and K) qRT-PCR analysis of IL-1β and IL-6 mRNA expression in macrophages post-transfection with miR-210-3p mimics and/or transduction with Lv-KLF7 ( n = 6). (L and M) Representative immunofluorescence images (L) and quantification (M) of KLF7 expression in RAW264.7 macrophages transfected with miR-210-3p mimics and transduced with Lv-KLF7 (KLF7, green; nuclei, blue [DAPI]; n = 4; scale bars, 50 μm). (N and O) Representative DCFH-DA fluorescence images (N) and quantification (O) of intracellular ROS levels (green). Red fluorescence (mCherry) indicates macrophages successfully transduced with the lentiviral vectors. Note that KLF7 overexpression attenuates miR-210-3p-induced oxidative stress. (scale bars, 50 μm; n = 6). (P and Q) Representative BODIPY-cholesterol fluorescence images (P) and quantification of intracellular fluorescence intensity (Q) in macrophages transfected with miR-210-3p mimics and transduced with Lv-KLF7 ( n = 3; scale bars, 50 μm). (R and S) Representative oil red O staining (R) and semi-quantitative analysis of oil red O-positive lipid area (S) in macrophages transfected with miR-210-3p mimics and transduced with Lv-KLF7 ( n = 5; scale bars, 100 μm). Values are shown as mean ± SEM. Two-group comparisons were performed using unpaired, two-tailed Student’s t tests, and multi-group comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple-comparisons post-hoc test. Sample sizes (n) indicate biological replicates per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5705/pmc12995705/pmc12995705__gr6.jpg)
![(a) Gene expression heatmap illustrates the relative expression levels of key genes (detected by real-time PCR) across all the six groups [the control, oxygen and glucose deprivation/reoxygenation (OGD/R), OGD/R+ negative control (NC), OGD/R+CLIC1 silencing, OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition, and OGD/R+CLIC1 silencing+NLRP3 activation]. Each row represents a specific gene, and each column represents a sample. (b) Correlation heatmap showing the Spearman correlation coefficients among the genes detected by real-time PCR. Each cell represents the correlation between two genes, with the colour gradient indicating the strength and direction of the correlation. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5528/pmc12445528/pmc12445528__pone.0332698.g008.jpg)