Abstract
The full repertoire of human microRNAs (miRNAs) that could distinguish common (benign) nevi from cutaneous (malignant) melanomas remains to be established. In an effort to gain further insight into the role of miRNAs in melanoma, we applied Illumina next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of nevi, thick primary (>4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines (both primary and metastatic) (n = 28). From this data representing 698 known miRNAs, we defined a set of top-40 list, which properly classified normal from cancer; also confirming 23 (58%) previously discovered miRNAs while introducing an additional 17 (42%) known and top-15 putative novel candidate miRNAs deregulated during melanoma progression. Surprisingly, the miRNA signature distinguishing specimens of melanoma from nevus was significantly different than that of melanoma cell lines from melanocytes. Among the top list, miR-203, miR-204-5p, miR-205-5p, miR-211-5p, miR-23b-3p, miR-26a-5p and miR-26b-5p were decreased in melanomas vs. nevi. In a validation cohort (n = 101), we verified the NGS results by qRT-PCR and showed that receiver-operating characteristic curves for miR-211-5p expression accurately discriminated invasive melanoma (AUC = 0.933), melanoma in situ (AUC = 0.933) and dysplastic (atypical) nevi (AUC = 0.951) from common nevi. Target prediction analysis of co-transcribed miRNAs showed a cooperative regulation of key elements in the MAPK signaling pathway. Furthermore, we found extensive sequence variations (isomiRs) and other non-coding small RNAs revealing a complex melanoma transcriptome. Deep-sequencing small RNAs directly from clinically defined specimens provides a robust strategy to improve melanoma diagnostics.
Highlights
The incidence and mortality of melanoma have continually increased over the past decades in the US
Increasing incidence is coupled by diagnostic discrepancies [2] whereby a considerable number of clinically suspicious pigmented tumors may show ambiguous histopathology making the classification between benign and malignant melanocytic tumors difficult and the clinical behavior unpredictable [3]
Clinical follow-up and melanoma staging were documented according to the American Joint Committee on Cancer (AJCC) (Table 1)
Summary
The incidence and mortality of melanoma have continually increased over the past decades in the US. A growing number of studies have shown select miRNAs deregulated in melanoma using mostly cell lines [11,12,13,14,15,16,17] and a few using specimens of melanoma metastases [18] or primary tumors [19,20,21,22] based on microarray hybridization or real-time quantitative RT-PCR (qRT-PCR) These methods feature reliable clustering of melanomas by similar miRNA expression profiles, they are limited to the detection of known miRNA sequences previously identified by sequencing or homology searches [23]. Alternative Sanger sequencing for miRNA profiling in archived melanoma and nodal specimens [24], while useful in detecting known and novel sequences, were initially complex and expensive due to laborious cloning techniques [25], have become practical due to the development of nextgeneration sequencing (NGS)
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