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miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) <t>Mitophagy</t> levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.
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miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) <t>Mitophagy</t> levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.
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miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) <t>Mitophagy</t> levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.
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miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) <t>Mitophagy</t> levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.
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miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) <t>Mitophagy</t> levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.
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miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) Mitophagy levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.

Journal: Frontiers in Immunology

Article Title: METTL3-mediated m6A modification regulates D-galactose-induced skin fibroblast senescence through miR-208a-5p

doi: 10.3389/fimmu.2025.1577783

Figure Lengend Snippet: miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) Mitophagy levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.

Article Snippet: After treatment, the mitophagy of MSFs was assessed using the Mitophagy Detection Kit (Dojindo Molecular Technologies, Inc., Japan), following the manufacturer’s instructions.

Techniques: Binding Assay, Luciferase, Reporter Assay, Western Blot, Expressing, Transfection, Knockdown, Staining