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. 2020 Jun 24;21:492–511. doi: 10.1016/j.omtn.2020.06.015

Figure 5.

Figure 5

Sal-miR-58 Induces VSMC Autophagy by Relieving KLF3 Repression of NEDD4L Expression

(A) Schematic representation of the −1880 to +34 bp region of the NEDD4L promoter and primers for amplifying the different regions of the NEDD4L promoter. ChIP analysis of KLF3 binding to the NEDD4L promoter was performed in VSMCs transfected with pcDNA3.1-KLF3. Data are the mean ± SEM (n = 3). ∗∗∗p < 0.001 versus pcDNA3.1. (B) A luciferase reporter controlled by the NEDD4L promoter was transfected into 293A cells along with pcDNA3.1 or pcDNA3.1-KLF3. Luciferase activity was measured using the dual luciferase reporter assay system. Data represent the relative NEDD4L promoter activity normalized to pRL-TK activity. ∗∗∗p < 0.001 versus pGL3-basic. (C) VSMCs were treated with Ang II or Sal-miR-58 (100 nM) alone or together, and the total RNA was harvested and analyzed by qRT-PCR. ∗p < 0.05 versus 0 M Ang II or miR-Ctl; #p < 0.05 versus 0 M AngII+Sal-miR-58. (D) VSMCs were treated as in (C), and the protein was harvested and analyzed by western blotting using anti-KLF3 or anti-NEDD4L antibodies. (E) Expression of KLF3 and NEDD4L in VSMCs treated as in (C) was examined by immunofluorescence staining. Green, red, and blue staining indicates NEDD4L, KLF3, and the nuclei, respectively. Scale bar, 100 μm. (F and G) VSMCs were transfected with Sal-miR-58 or pcDNA3.1-KLF3 alone or together for 24 h, and expression of KLF3 and NEDD4L was determined by (F) qRT-PCR and (G) western blot analysis. ∗p < 0.05 versus pcDNA3.1; #p < 0.05 versus pcDNA3.1+Sal-miR-58. (H) VSMCs were transfected with pcDNA3.1-NEDD4L and treated with/without Ang II, the total RNA was harvested, and Atg5, Beclin1, and LC3B expression levels were analyzed by qRT-PCR. ∗p < 0.05, ∗∗p < 0.01 versus pcDNA3.1. (I) VSMCs were treated as in (H), and the protein was harvested and analyzed by western blotting using anti-Beclin1, anti-Atg5, and anti-LC3B. (J) VSMCs were treated as in (H) and infected with RFP-GFP-LC3B double-labeled adeno-associated virus, and autophagosomes and autolysosomes were examined as described above. Scale bar, 100 μm. (K) VSMCs were transfected with Sal-miR-58 or si-NEDD4L alone or together for 24 h, and expression of Beclin1, Atg5, and LC3B was determined by western blotting. (L) VSMCs were treated as in (K) and infected with RFP-GFP-LC3B double-labeled adeno-associated virus, and autophagosomes and autolysosomes were examined as described above. Scale bar, 100 μm.