In an unbiased approach to identify
‘melanoma-specific’ microRNAs (miRNAs) that are relevant to melanomagenesis, we
used miRNA microarrays (miRBaseV18) to comprehensively study the miRNA profile
of cutaneous melanoma (n=55) in relation to other solid malignancies (n=34).
Among 233 differentially expressed (≥2 fold, p.≤0.05) miRNAs, we found miR-514a
to be upregulated (mean 204 fold) in melanoma cell lines (38/55, 69%) compared
with other solid cancers (3%, 1/34). miR-514a is a member of a cluster
(miR-506-514) that has been shown previously to be involved in initiating
melanocyte transformation along with promotion of melanoma growth. As no
gene-specific targets had been identified, we used another unbiased approach to
identify true binding targets of miR-514a. Biotin-labelled miR-514a ‘pull-down’
data revealed hundreds of genes that have a known association with melanoma,
including CTNNB1, CDK2, MC1R, and NF1. The latter,
a well known melanoma tumour suppressor gene, was selected for functional
validation as it has recently been implicated in resistance to current
RAF-targeted therapies in patients harbouring BRAFV600E mutant tumours. NF1 was
confirmed to be a direct target of miR-514a using a luciferase-reporter assay
following site-directed mutagenesis of predicted miR-514a binding sites.
Moreover, induction and inhibition of miR-514a into melanoma cell lines modified
NF1 levels. We next sought to investigate the effect of miR-514a expression in
relation to PLX4032 inhibition of BRAFV600E mutant melanoma cell lines. In
cytotoxicity assays, we showed that modifying miR-514a levels alters proliferation
rates in BRAFV600E cells treated with PLX4032. These data provide yet another
mechanism for the dysregulation of the MAPK pathway which helps explain the
profound resistance to current RAF-targeted therapy.