Abstract
miRNAs are key regulatory small non-coding RNAs involved in critical steps of melanoma tumorigenesis; however, the relationship between sequence specific variations at the 5′ or 3′ termini (isomiR) of a miRNA and cancer phenotype remains unclear. Deep-sequencing and qRT-PCR showed reduced expression of miR-144/451a cluster and most abundant isomiR (miR451a.1) in dysplastic nevi, in-situ and invasive melanomas compared to common nevi and normal skin (n = 101). miRNA in situ hybridization reproducibly confirmed lost miR-451a.1 in melanoma compared to nevus cells or adjacent keratinocytes. Significantly higher expression of miR-451a.1 was associated with amelanotic phenotype in melanomas (n = 47). In contrast, miR-451a was associated with melanotic phenotype, absent pagetoid scatter of intraepidermal melanocytes, superficial spreading histological subtype and tumor inflammation. Sequencing miRNAs from cultured melanocytes with cytoplasmic melanin gradient (light, medium to dark) showed absent miR-451a while revealing other melanin-associated miRNAs, e.g. miR-30b, miR-100 and miR-590 in darkly and let-7a, let-7i and let-7f in lightly to moderately pigmented cultured melanocytes. Ectopic expression of miR-144/451a in melanoma cell lines resulted in markedly higher levels of mature miR-451a.1 than miR451a or miR-144; and significantly retarded cell migration and inhibited invasion in a glucose-sensitive manner. Surprisingly, these effects were not mediated by calcium binding protein 39 (CAB39), a proven miR451a gene target. miR-144/miR-451a cluster is a novel miRNA locus with tumor suppressive activity in melanoma.
Highlights
The incidence and mortality of melanoma have continually increased over the past decades in the US
Down-regulation of miR-144/451a in melanoma We applied next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of common nevi (CN) and primary cutaneous melanomas (PCM) and defined a set of top-40 list, which properly classified normal from cancer [14]
MiR-144/451a cluster ranked among the top-40 miRNAs and showed a 3-fold reduction (% per total miRNA) of miR451a and miR-144-3p in PCM compared to NS; and absent from melanocytes and melanoma cell lines (Table S1 in File S1)
Summary
The incidence and mortality of melanoma have continually increased over the past decades in the US. We established a full repertoire of human miRNA transcriptome by deep-sequencing small non-coding RNAs (18– 30 nt) directly in biopsies of melanocytic nevi (benign melanocytic hyperplasia), thick primary (.4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines [14]. These results defined a set of top-40 miRNAs, which properly classified nevus from melanoma, and demonstrated extensive sequence variations (isomiRs), revealing a higher complexity of different miRNA populations in melanoma that may participate in clinically relevant gene regulatory networks [14]. It has been demonstrated that isomiRs are not artifacts generated from deep sequencing, rather mature variants that result in different cleavage sites for RNASEN and Dicer [17], enzymes in miRNA biogenesis pathway. isomiRs
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