miR-7 overexpression promotes A549 cell proliferation through negatively regulating KLF4.

<p>(A) 2×10⁴ cells of pcDNA, miR-7 and miR-7+KLF4 overexpressing clones were plated in 24 well culture plates. Cells were counted every 24 hours during four consecutive days. (B) Once A549 cells reached confluence (T = 0), cell numbers were determined each 24 hours during two consecutive days. Fresh medium was added 24 hours post-confluence. Results represent the mean of six independent experiments performed with five or six pcDNA, miR-7 or miR-7+KLF4 overexpressing independent clones. ***<i>p</i><0.001, *<i>p</i><0.05 <i>vs.</i> pcDNA. (C) Protein levels of KLF4 target genes products. pcDNA and miR-7 expressing A549 cells were harvested at 0, 24 and 48 hours post-arrest, total cell extracts were prepared and Cyclin D and p27 protein levels were determined by Western blot analysis. ERK2 protein was used as loading control. Numbers denote the protein levels as the percentage of each condition relative to the pcDNA cells at 0 hours post-arrest. A representative Western blot of three independent experiments is shown.