In-Depth Characterization of microRNA Transcriptome in Melanoma

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.

Identification of microRNAs associated with invasive and aggressive phenotype in cutaneous melanoma by next-generation sequencing

“…18 To decrease the chance of sequencing unwanted ribosomal RNAs, 'poison primers' were added to the ligated products. 27 Briefly, after reverse transcription, a cDNA library of mixed barcodes was generated for Illumina genome analyzer (IGA) II sequencing.…”

“…miRDeep 2.0 Analysis RNA sequencing data in FASTQ form was input into the microRNA-characterization software miRDeep 2.0 on a Linux platform as previously described. 18 Briefly, the software used Bowtie to map the reads to the UCSC reference genome GRCh37 (browser hg19), allowing for only one mismatch and compiling stretches of hairpin sequences up to 140 nt as microRNA precursors. The precursors are then folded into two-dimensional structures using RNAfold from Vienna RNA Package 1.8.5. miRDeep 2.0 evaluated structure and identified miRNAs based on: minimal free energy, length of the 3′ overhang, existing homologs, distribution of passenger (star), guide (mature), loop sequences, and frequency of the predominant read within the sequencing data.…”

“…14 Next-generation sequencing of small RNAs isolated from common melanocytic nevi, thick primary melanomas (44.0 mm in invasive depth) and metastatic melanomas to skin and lymph nodes revealed a top 40 list of key miRNAs that correctly distinguished benign from malignant melanocytic tumors. 18 Among them was miR-211 whose expression was significantly decreased in melanomas compared with common nevi in an independent, validation cohort. 18 miR-211 functions as a metabolic switch 19 and as a potent tumor suppressor in vitro influencing gene pathways involved in cell invasion.…”

“…18 Among them was miR-211 whose expression was significantly decreased in melanomas compared with common nevi in an independent, validation cohort. 18 miR-211 functions as a metabolic switch 19 and as a potent tumor suppressor in vitro influencing gene pathways involved in cell invasion. [20][21][22] It is located within the noncoding sequence (intron 6) of Melastatin-1 (MLSN-1) gene, 20 which is expressed at high levels in melanocytic nevi but significantly reduced in metastatic melanomas with variable expression in primary lesions.…”

Five miRNAs as Novel Diagnostic Biomarker Candidates for Primary Nasopharyngeal Carcinoma

“…Nevertheless, the role of miR-205 remains controversial. For example, the downregulation of miR-205 is useful in distinguished melanoma from nevus (Kozubek et al, 2013). Moreover, the relative expression of miR-205-5p in any Gleason pattern was decreased significantly compared with normal tissues (Tsuchiyama et al, 2013), to include prostate cancer samples compared with non-prostate cancer samples (Larne et al, 2013).…”