Figure 5
MiR-449a inhibits cell proliferation and arrests the cell cycle by targeting multiple genes.
HepG2.2.15 cells were transfected with 20 nM miR-449a. After 24 h, the cells were split at a density of 10,000/well to perform the following experiments. (A) Cells were distributed into 96-well plates and cell growth was measured every 24 h for 5 days using the WST-1 assay. (B) Cells were serum-starved overnight for 48 hours, followed by the addition of serum and 3H-thymidine; incorporation of 3H-thymidine into cellular DNA after 4 h was measured using a scintillation counter. (C) Cells were treated with nocodazole (100 nM), aphidicolin (4 μg/ml) or medium control for 24 h. The distribution of cells in the cell cycle phases G1, S and G2/M was assessed by flow cytometry using propidium iodide staining and the percentage of cells in the different phases of the cell cycle is shown. Each experiment was performed in triplicate with two different batches of transfected cells. (D) HepG2.2.15 cells were transfected with different concentrations of miR-449a for 4 days. E2F1, CDK6, HDAC1, ERK1/2, phosphorylated-Rb and-ERK1/2 protein levels were measured by western blotting with specific antibodies and normalized to β-actin as a loading control. *P < 0.05.
