Figure 5. Biological consequences of miR-210, miR-147a and miR-147b overexpression on A549 cells proliferation and viability.

A549 cells were transfected with 10 nM pre-miR-210, pre-miR-147a, pre-miR-147b or pre-miR-Neg and analyzed for several proliferation (A-E) and viability (F-G) parameters. A) Confluent cell monolayer was wound and filmed for 55h under light time laps microscope. Curves represent wound beds closure quantified by measuring the wound bed surface at the indicated times after injury using the Image J software. Values are expressed in percentage of the initial surface and correspond to the mean ± SD of 3 microscope fields. B) Effect of miR-210 and miR-147 family on A549 cell proliferation. Exponentially growing A549 cells were transfected and counted each day during 4 days with blue Trypan. Data show mean ± SD values of trypan blue negative (left panel) and trypan-blue positive cell number (right panel) from 2 independent experiments performed in triplicate. C) Cells were stained with propidium iodide and analyzed by flow cytometry. The G0/G1 (1), S (2) and G2/M (3) fractions were quantified in each condition. D) Quantification of each of these 3 fractions (G0/G1, S and G2/M) from 3 independent experiments. E) Expression of Cyclin D, Cyclin A, Cyclin E, CDK4, CDK6, pRB (6 molecules involved in G1 phase progression), p27Kip1 (inhibitor of G1 phase progression) and Cyclin B (involved in G2/M phase) were assessed by Western blot. Hsp60 corresponds to the loading control. F) Caspase 3/7 assay was performed at 3, 4 and 5 days after transfection. Data are mean ± SD values of 3 independent experiments performed in triplicate. See also Figure S4A. G) Expression of active caspase-3 (cleaved) and PARP, a substrate of caspase-3 was analyzed by Western blot. HSP60 corresponds to the loading control. Normalized densitometric quantification are shown for each lane. See also Figure S4C and Figure S4D (*p<0.05, **p<0.005, ***p<0.0005).