Figure 9
Effect of miR15a suppression in normal cholangiocytes on miR15a levels, Cdc25A expression, cell proliferation, and cyst growth.
(A) Representative light (top panels) and fluorescent (bottom panels) images show that both scrambled miRNA and anti-miR15a were present in cultured cells in contrast with cholangiocytes treated with vehicle. Original magnification, ×20. (B) Quantitative PCR demonstrates that levels of miR15a in cholangiocytes treated with anti-miR15a (anti) were decreased compared with those in cholangiocytes treated with vehicle or scrambled miRNA. Data are representative of 3 independent experiments. (C) Representative Western blot and quantitative analysis based on densitometry measurements (D) of 5 independent experiments show that in anti-miR15a cholangiocytes, Cdc25A protein levels were increased by 38%. Thin white lines on the gel indicate that the lanes were run on the same gel but were noncontiguous. (E) Rates of cell proliferation were assessed in cholangiocytes treated with scrambled miRNA (n = 5) or anti-miR15a (n = 6) and expressed as a percentage of changes compared with rates of proliferation in nontreated cholangiocytes (considered to be equal to 100%). Cholangiocytes exposed to miR15a antisense display higher rates of proliferation than cholangiocytes exposed to scrambled miRNA. (F) Representative light microscopic images of cystic structures formed in 3D culture by normal cholangiocytes and quantitative assessment of changes in cystic areas over time (G) reveal that in the presence of anti-miR15a, cysts grow more rapidly compared with those in the presence of scrambled miRNA. Changes shown in G are in comparison with day 0. *P < 0.05. Scale bars: 250 μm.
