Figure 5.

Functional validation of BRAFV600E-X1 binding microRNAs that do not alter BRAFV600E-X1 mRNA levels and rather affect its translation (Class III).

(a). Luciferase assay performed in HCT116 Dicer-/- cells indicates that miR-619-5p, miR-1260a, miR-1260b, miR-7704, let-7b-5p, let-7e-5p and let-7i-5p are functional, as they cause a decrease (miR-619-5p, miR-7704, let-7b-5p, let-7e-5p and let-7i-5p, Class IIIa) or an increase (miR-1260a and miR-1260b, Class IIIb) in reporter gene activity.

(b-c). Quantification of BRAFV600E (b) and pMEK (c) protein levels, as detected by immunoblot 48 h after the transfection of si-miR-619-5p, si-miR-1260a, si-miR-1260b, si-miR-7704, si-let-7b-5p, si-let-7e-5p and si-let-7i-5p into A375 melanoma cells.

(d). Compared to si-NC, the Relative Translation Efficiency (RTE) of Luciferase-X1-3ʹUTR chimerical construct, which is the ratio between Luciferase protein activity and Luciferase mRNA level [28], is lower in presence of si-miR-619-5p and si-let-7b-5p and higher in presence of si-miR-1260a.

(e). Effect of the overexpression of miR-619-5p and let-7b-5p on the proliferation of A375 melanoma cells and dependency on BRAFV600E-X1. Compared to si-NC, the transfection of si-miR-619-5p and si-let-7b-5p causes a net decrease in cell proliferation. Such effect is partially abrogated by the concomitant overexpression of the microRNA-insensitive BRAFV600E-X1 CDS, which indicates that the microRNA acts, at least in part, by targeting of this protein. CTRL: A375 cells stably infected with the empty pCW lentiviral vector, transfected with si-miR-619-5p or si-let-7b-5p and induced with 2 ug/ml doxycycline for 48 h. V600E-X1: A375 cells stably infected with pCW-BRAFV600E-X1-CDS lentiviral vector, transfected with si-miR-619-5p or si-let-7b-5p and induced with 2 ug/ml doxycycline for 48 h. The proliferation of A375 cells stably infected with pCW-BRAFV600E-X1-CDS lentiviral vector, transfected with si-NC and induced with 2 ug/ml doxycycline for 48 h did not show a statistically significant difference compared to the proliferation of A375 cells stably infected with the empty pCW lentiviral vector, transfected with si-NC and induced with 2 ug/ml doxycycline for 48 h (which was taken as reference for all the other experimental conditions).

(f). Effect of the overexpression of miR-619-5p and let-7b-5p on colony forming ability of A375 melanoma cells.

(g). Effect of the overexpression of miR-619-5p and let-7b-5p on the growth of A375-mCherry cells xenografted into zebrafish embryos. About 48 h after the transfection with si-miR-619-5p or si-let-7b-5p, the cells were injected in 48 h post fertilization embryos and allowed to grow for an additional 48 h. At the end of this period, the size of red cell masses was measured. The pictures are taken from one out of three independent experiments performed, all with comparable outcome. Scale bar: 100 um. The graphs in this figure represent the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

(h). In the skin cutaneous melanoma dataset available at TCGA, patients with higher levels of let-7b-5p (above the mean, red) show a better overall survival than patients with lower levels of miR-1246 (below the mean, green). n = 501. Samples above the mean: 287, 116 events. Patients below the mean: 214, 96 events. p = 0.0149.

(i). Cartoon summarizing the mechanism of action of the X1-targeting microRNAs identified in this article and their effect on the biological properties of melanoma cells.