Characterization of the Melanoma miRNAome by Deep Sequencing

Abstract

Background: MicroRNAs (miRNAs) are 18-23 nucleotide non-coding RNAs that regulate gene expression in a sequence specific manner. Little is known about the repertoire and function of miRNAs in melanoma or the melanocytic lineage. We therefore undertook a comprehensive analysis of the miRNAome in a diverse range of pigment cells including: melanoblasts, melanocytes, congenital nevocytes, acral, mucosal, cutaneous and uveal melanoma cells.

Methodology/principal findings: We sequenced 12 small RNA libraries using Illumina's Genome Analyzer II platform. This massively parallel sequencing approach of a diverse set of melanoma and pigment cell libraries revealed a total of 539 known mature and mature-star sequences, along with the prediction of 279 novel miRNA candidates, of which 109 were common to 2 or more libraries and 3 were present in all libraries.

Conclusions/significance: Some of the novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers to assist in the early detection of distant metastases by measuring the circulating levels in blood. Follow up studies of the functional roles of these pigment cell miRNAs and the identification of the targets should shed further light on the development and progression of melanoma.

Figure 1. Flowchart representing the steps involved to annotate known microRNAs and the discovery of predicted candidate microRNAs using miRanalyzer and CID-miRNA .

White boxes represent the range of numbers for an individual library. Green boxes represent the total number of unique miRNAs across all libraries. Blue boxes represent unmatched reads which were not considered further.

Figure 2. Example of a mature miRNA (hsa-mir-29a) showing variations in the 5′ and 3′ ends.

The optimal secondary structure is represented in dot-bracket notation (where brackets and full-stops represent complementary and non-complementary nucleotides respectively) with the published mature miRNA bolded.

Figure 3. Unsupervised hierarchical clustering using the Pearson's correlation between all expressed miRNAs.

QF1160MB, melanoblasts; MELB, melanocytes; D10, acral melanoma; D11, mucosal melanoma; MEL202, uveal melanoma.

Figure 4. Example of sequence conservation in a known miRNA (hsa-mir-1287) and a novel candidate miRNA (MELmiRNA_293).