A functional screen identifies specific microRNAs capable of inhibiting human melanoma cell viability

Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy

Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma

Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic

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

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

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

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