Figure 4.

Sal-miR-58 Inhibits Ang II-Induced Inflammation by Downregulating KLF3 in VSMCs

(A) mRNAs for KLF3, KLF4, and KLF5 were determined by qRT-PCR in VSMCs transfected with Sal-miR-58. ^nsp > 0.05 versus miR-Ctl (n = 3). ns, not significant. (B) Western blot analysis of KLF3, KLF4, and KLF5 in VSMCs transfected with Sal-miR-58 or miR-Ctl. (C) Immunofluorescence staining of KLF3 (red) and the nucleus (DAPI; blue) in VSMCs treated with Sal-miR-58 or Sal-miR-58+Ang II. Scale bar, 100 μm. (D) Luciferase reporter assays were performed in HEK293 cells co-transfected with Sal-miR-58 and a wild-type or mutant KLF3 3′ UTR-luciferase reporter. ∗p < 0.05 versus miR-Ctl; ^##p < 0.01 versus Sal-miR-58+wild-type. The Sal-miR-58-binding site is shown in green; the mutated sites are shown in red. (E) Relative expression of KLF3 mRNA was examined by qRT-PCR in VSMCs treated with different doses of Ang II and presented after normalizing to 18S rRNA (mean ± SEM; n = 3). ∗p < 0.05 versus 0 M Ang II. (F) VSMCs were treated with different doses of Ang II for 24 h, and expression of KLF3 was examined by western blotting. (G) Expression of KLF3 in Ang II-treated VSMCs was examined by immunofluorescence staining. Red and blue stainings indicate KLF3 and the nuclei, respectively. Scale bar, 100 μm. (H) VSMCs were treated with different doses of Ang II for 24 h, and expression of KLF3 in the nucleus and the cytoplasm was examined by western blotting. (I) VSMCs were transfected with Sal-miR-58 or pcDNA3.1-KLF3 alone or together for 24 h, and expression of NF-κB p50 and p65 was determined by western blotting. (J) VSMCs were transfected as in (I), and levels of IL-1β, IL-6, and TNF-α in the culture medium were determined by ELISA. ∗∗p < 0.01, ∗∗∗p < 0.001 versus pcDNA3.1; ^#p < 0.05 versus pcDNA3.1-KLF3.