A genome-wide screen for miRNAs that inhibit A375 melanoma cell growth.

<p>(A) Inhibition of melanoma growth was measured by means of cell viability and cell count. For each sample a B-score was calculated and B-scores from both assays are plotted against each other. There is a strong correlation between both assays. The B-scores for one miRNA, miR-518b, fell outside the range of the graph: they were −6 for cell count and −12 for cell viability. (B) A comparison with a normal distribution shows that the cell viability screen is sensitive for identifying growth-inhibitory miRNAs. A concomitant estimate of the false discovery rate is shown in grey fill (secondary axis). (C) 55 potential inhibitory miRNAs were tested in a confirmation screen against 11 empty vector samples and a population of 28 miRNAs with small or no effects in the primary screen. Box plots show values between 25^th and 75^th percentile in boxes, and the outermost values as whiskers. 20 of 55 inhibitory miRNAs scored better than any of the control miRNAs (below dashed line). *p = 6.8*10⁻⁵, **p = 1.6*10⁻⁶, ***p = 5.4*10⁻¹⁰. (D) Individual hits selected for follow-up, and their relative effect on cell viability. A virus containing a short-hairpin construct targeting BRAF was used as a positive control. Error bars represent standard deviation of three samples.</p

Transcriptome analysis after miR-203 transfection.

<p>(A) A375 cells were transfected with either miR-203 or scrambled control and the transcriptome was quantified by RNA-Seq. All differentially expressed genes are plotted in the left graph, while only the differentially expressed genes containing miR-203 target sites are plotted in the right graph. Genes with miR-203 target sites are much more likely to be downregulated after miR-203 overexpression, and downregulated genes are highly enriched for genes with miR-203 target sites (p<0.0001). (B) One of the differentially expressed genes after miR-203 transfection is BIRC5. Repression was examined at both the mRNA and the protein level by qPCR (left) and Western blot (right) respectively. The BIRC5 transcript and its protein product survivin are both reduced after miR-203 transfection, but also after siBRAF transfection.</p

Endogenous miRNA expression and expression after lentiviral transduction in A375 cells.

<p>qPCR data are relative to U6 small RNA. Endogenous: empty vector-transduced, ectopic: miRNA-transduced.</p

Effect of introduction of synthetic miRNAs on A375 viability.

<p>A375 cells were transfected with a range of concentrations of different miRNAs, and 72 hours after transfection viability was measured by means of MTS assay. Effects are compared to a scrambled control and a pool of 4 siRNAs against BRAF (siBRAF) as a positive control for A375 growth inhibition. Specific effects of miRNAs are best observed at concentrations of 10 nM. Each panel shows a different subset of miRNA mimics, although miRNAs were assessed in the same experiment. Error bars represent standard deviation of three samples. A representative of three experiments is shown.</p

Comparison of miRNA-induced effects in several melanoma cell lines.

<p>Cells were transfected with 10 nM (A375 and A2058) or 30 nM (SK-MEL-28 and SK-MEL-173) RNA and cell viability was measured 72 hours after transfection. Data are plotted relative to a mock-infected control. Error bars represent standard deviation of three samples.</p