(A) qRT-PCR of c-Jun mRNA showed an opposite effect of the two microRNAs, miR-200a and miR-200b, on the expression level of endogenous c-Jun mRNA. The mean value of the corresponding empty vector was set to one, and data represent the mean ± SD of two independent experiments. (B) JUN protein level measured by Western blotting. The graphic on the left represents the densitometry analysis of three independent experiments; on the right there is only one representative WB experiment; the empty vector was set to one. (C) Inhibition of transcription by actinomycin D. HEK 293T cells were transfected with the miR-200a overexpressing plasmid or with the empty vector. Twenty-four hours after transfection, cells were treated with actinomycin D for the times indicated (bottom of the graph). Total mRNA was extracted, quantified, and analyzed by qRT-PCR with primers detecting the endogenous c-Jun mRNA. The amount of c-Jun mRNA present in the cells, transfected with miR-200a overexpressing plasmid or the empty vector control, before the addition of actinomycin D was utilized as reference point and set to one. (D) qRT-PCR of c-Jun, matrix metallopeptidase 9 (Mmp-9), and vascular endothelial growth factor A (Vegf-A) mRNAs performed with RNA from MCF7 cells transfected with LNA microRNA inhibitor anti-miR-200a, miR-200b, or scramble. (E) (Left panel) Wound healing assay with metastatic breast cancer cells line, MDA-MB-231. Cells were transfected with empty vector or miR-200b and scramble or c-Jun siRNA. Twenty-four hours after transfection we generated a scratch in the cells' monolayer, and we observed the cells migration rate hourly, for 24 h. The white bar in the panels represents 250 µm. (Right panel) Western blotting evaluation of siRNA knockdown. Proteins were isolated after wound healing assay and actin serves as loading control. Data represent the mean ± SD, and asterisks (*) in all the figures indicate statistically significant modulations with respect to empty vector according to paired Student's test. (*) P < 0.05; (**) P < 0.01.
