A comprehensive repertoire of human microRNAs (miRNAs) that could be involved in early melanoma invasion into the dermis remains unknown. To this end, we sequenced small RNAs (18-30 nucleotides) isolated from an annotated series of invasive melanomas (average invasive depth, 2.0 mm), common melanocytic nevi, and matched normal skin (n = 28). Our previously established bioinformatics pipeline identified 765 distinct mature known miRNAs and defined a set of top 40 list that clearly segregated melanomas into thin (0.75 mm) and thick (2.7 mm) groups. Among the top, miR-21-5p, let-7b-5p, let-7a-5p, miR-424-5p, miR-423-5p, miR-21-3p, miR-199b-5p, miR-182-5p, and miR-205-5p were differentially expressed between thin and thick melanomas. In a validation cohort (n = 167), measured expression of miR-21-5p and miR-424-5p, not previously reported in melanoma, were significantly increased in invasive compared with in situ melanomas (Po0.0001). Increased miR-21-5p levels were significantly associated with invasive depth (P = 0.038), tumor mitotic index (P = 0.038), lymphovascular invasion (P = 0.0036), and AJCC stage (P = 0.038). In contrast, let-7b levels were significantly decreased in invasive and in situ melanomas compared with common and dysplastic nevi (Po0.0001). Decreased let-7b levels were significantly associated with invasive depth (P = 0.011), Clark's level (P = 0.013), ulceration (P = 0.0043), and AJCC stage (P = 0.011). These results define a distinct set of miRNAs associated with invasive and aggressive melanoma phenotype. Notwithstanding the distinct sets of DNA and chromosomal alterations demonstrated in melanoma, the changes in the noncoding RNA transcriptome remain poorly understood. The microRNAs (miRNAs) are endogenous, noncoding small (18-24 nucleotides) RNAs, which can regulate gene expression in animals and plants by complementary base-pairing to the mRNAs of target genes to specify mRNA cleavage or translation repression. 1 Growing evidence has shown that particular miRNAs function predominately as tumor suppressors, eg, let-7 family 2,3 and miR-15a and miR-16; 4 and some as oncogenes, eg, miR-17~92 cluster. 5,6 A number of studies have demonstrated an evolving role of miRNAs in various aspects of melanoma biology and clinical behavior. For example, results showed a cell-specific upregulated Dicer expression in 81% of cutaneous, 80% of acrolentiginous, and 96% of metastatic melanomas compared with carcinomas or sarcomas of the skin. 7 Moreover, the expression of Dicer was significantly higher in melanomas compared with benign melanocytic nevi. Dicer, a member of the RNase III family of double-stranded RNases, is a central enzyme in a multi-component miRNA biogenesis pathway where the Drosha/DGCR8 complex and Dicer act sequentially to crop long primary and precursor miRNAs into functionally mature miRNAs. 8 In patients with cutaneous melanomas, Dicer upregulation was significantly associated with an increased tumor mitotic index, Breslow's depth of invasion, nodal metastasis, and a higher American Joi...
Some melanocytic tumors can be histologically ambiguous causing diagnostic difficulty, which could lead to overtreatment of benign lesions with an unwarranted psychological distress or undertreatment of malignant cancers. Previously, we demonstrated that significantly decreased miR-211 expression in melanomas compared with melanocytic nevi could accurately discriminate malignant from benign tumors. Herein we show microRNA in situ hybridization for fluorescent detection of miR-211, suitable for paraffin-embedded tissues in 109 primary melanocytic tumors. miR-211 expression was significantly lower in melanomas vs nevi (P<0.0001), and receiver operating characteristic curve (area under the curve=0.862) accurately discriminated melanomas from nevi with 90% sensitivity and 86.2% specificity. Qualitatively, all dysplastic nevi expressed miR-211 at high (86%) and low (14%) levels, independent of the degree of nuclear atypia. All 35 melanocytic tumors with Spitz morphology expressed miR-211 independent of morphological classification, where clinical follow-up of these patients showed no recurrence at the site or metastasis in mean and median of 3 (ranging 2-5) years. Moreover, a decision tree learning analysis selected age and miR-211 miRNA in situ hybridization as the predictive variables for benign or malignant outcome in 88 patients correctly classified 92% (81 out of 88) of cases as proven by receiver operating characteristic curve (area under the curve=0.9029). These results support miR-211 as a leading miRNA candidate for melanoma diagnosis and miRNA in situ hybridization as a uniquely uncomplicated ancillary test.
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.
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