Figure 4.

FBXW7 Is a Direct Target Gene of miR-25

(A) A potential target site (highlighted in red) of miR-25 on the FRXW7 3′ UTR was predicted by TargetScan. The mutated target sequence is shown below. (B) A luciferase reporter assay showed that the predicted binding sequence was required for miR-25 inhibition (n = 3). (C) qRT-PCR showed that miR-25 overexpression decreased FBXW7 expression in hESC-CMs (n = 3). (D) Western blot analysis showed that miR-25 overexpression decreased FBXW7 expression in hESC-CMs. (E) Fold change expression of FBXW7 normalized by GAPDH as a internal control in hESC-CMs treated with miR-25 mimics or NC. (n = 3). (F) FBXW7 expression was knocked down by siFBXW7 in hESC-CMs (n = 3). (G) qRT-PCR showed that PCNA expression was significantly increased in hESC-CMs treated with siFBXW7 (n = 3). (H) EdU staining (green) revealed that FBXW7 knockdown increased CM proliferation. The number of EdU-positive cells is shown on the right. Nuclei were stained with DAPI (blue); CMs were stained with an antibody against α-ACTININ (red). Approximately 2,000 cells were quantified in each group. Scale bars, 150 μm. (I) Ki-67 staining (green) revealed that FBXW7 knockdown increased CM proliferation. The number of Ki-67-positive cells is shown on the right. Nuclei were stained with DAPI (blue); CMs were stained with an antibody against α-ACTININ (red). Approximately 2,000 cells were quantified in each group. Scale bars, 150 μm. Mut, mutant; siNC, siRNA negative control; WT, wild-type. Statistical significance was calculated using student's t test for paired samples. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.