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. Author manuscript; available in PMC: 2021 Sep 11.
Published in final edited form as: Circ Res. 2020 Jun 29;127(7):855–873. doi: 10.1161/CIRCRESAHA.120.316951

Figure 4. KD platelet-derived miR-223 regulates VSMC dedifferentiation via direct targeting PDGFRβ.

Figure 4.

VSMCs were incubated with platelets from HC and KD patients with NCAA for 48 hours, cell proliferation was assessed by, (A) CCK-8 assays (HC: n=17, KD (NCAA): n=16, Mann Whitney test), and (B) BrdU incorporation assays (HC: n=12, KD (NCAA): n=13, Unpaired t test). The expression (C) and quantification (D) of markers for differentiation (ACTA2, CNN1, TAGLN) and dedifferentiation (PCNA, OPN, PDGFRβ) in VSMCs co-cultured with platelets from HC (n=13) and KD patients with NCAA (n=21) were determined by Western blot (Mann Whitney test). Ago2 immunoprecipitation was performed in VSMCs (E) (Ctrl: n=6, KD (NCAA): n=6, Unpaired t test), or VSMCs pre-transfected with miR-223 inhibitor (antagomiR-223) (F) (KD (NCAA)+antagomiRNC: n=6, KD (NCAA)+antagomiR223: n=6, Mann Whitney test), after 24 hours incubation with platelets from KD patients with NCAA, Ago2-associated miR-223 and PDGFRβ mRNA were quantified by quantitative RT-PCR (RT-qPCR). VSMCs transfected with Luc-PDGFRβ (WT) or Luc-PDGFRβ (MT) were co-transfected with miR-223 mimic (agomiR-223) (n=3) or NC-mimic (agomiR-NC) (n=3) in (G), co-cultured with platelets from HC (n=3) or KD patients with NCAA (n=6) in (H), co-transfected with antagomiR-223 (n=6) or NC inhibitor (antagomiR-NC) (n=6) followed by incubation with platelets from KD patients with NCAA as indicated in (I). Luciferase activity was measured with that of Renilla as transfection control (Mann Whitney test or Unpaired t test). Abbreviations: HC, VSMCs co-cultured with platelets from HC; KD (NCAA), VSMCs co-cultured with platelets from KD patients with NCAA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.