Small noncoding microRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation or orchestrating their sequence-specific degradation. In this study, we investigated miRNA and miRNA target gene expression patterns in melanoma to identify candidate biomarkers for early and progressive disease. Because data presently available on miRNA expression in melanoma are inconsistent thus far, we applied several different miRNA detection and profiling techniques on a panel of 10 cell lines and 20 patient samples representing nevi and primary or metastatic melanoma. Expression of selected miRNAs was inconsistent when comparing cell line-derived and patient-derived data. Moreover, as expected, some discrepancies were also detected when miRNA microarray data were correlated with qPCR-measured expression levels. Nevertheless, we identified miRNA-200c to be consistently downregulated in melanocytes, melanoma cell lines, and patient samples, whereas miRNA-205 and miRNA-23b were markedly reduced only in patient samples. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Whole-genome microarrays were performed for analysis of selected melanoma cell lines to identify potential transcriptionally regulated miRNA target genes. Using Ingenuity pathway analysis, we identified a deregulated gene network centered around microphthalmia-associated transcription factor, a transcription factor known to play a key role in melanoma development. Our findings define miRNAs and miRNA target genes that offer candidate biomarkers in human melanoma. Cancer Res; 70(10); 4163-73. ©2010 AACR.

MicroRNAs (miRNAs) are ubiquitously expressed small non-coding RNAs that, in most cases, negatively regulate gene expression at the post-transcriptional level. miRNAs are involved in fine-tuning fundamental cellular processes such as proliferation, cell death and cell cycle control and are believed to confer robustness to biological responses. Here, we investigated simultaneously the transcriptional changes of miRNA and mRNA expression levels over time after activation of the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) pathway by interferon-γ stimulation of melanoma cells. To examine global miRNA and mRNA expression patterns, time-series microarray data were analysed. We observed delayed responses of miRNAs (after 24–48 h) with respect to mRNAs (12–24 h) and identified biological functions involved at each step of the cellular response. Inference of the upstream regulators allowed for identification of transcriptional regulators involved in cellular reactions to interferon-γ stimulation. Linking expression profiles of transcriptional regulators and miRNAs with their annotated functions, we demonstrate the dynamic interplay of miRNAs and upstream regulators with biological functions. Finally, our data revealed network motifs in the form of feed-forward loops involving transcriptional regulators, mRNAs and miRNAs. Additional information obtained from integrating time-series mRNA and miRNA data may represent an important step towards understanding the regulatory principles of gene expression.

BackgroundThe type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic cells. The role of IFN-γ in melanoma is not fully understood: it is a well-known growth inhibitor of melanoma cells in vitro. On the other hand, IFN-γ may also facilitate melanoma progression. While interferon-regulated genes encoding proteins have been intensively studied since decades, the contribution of miRNAs to effects mediated by interferons is an emerging area of research.We recently described a distinct and dynamic regulation of a whole panel of microRNAs (miRNAs) after IFN-γ-stimulation. The aim of this study was to analyze the transcriptional regulation of miR-29 family members in detail, identify potential interesting target genes and thus further elucidate a potential signaling pathway IFN-γ → Jak→ P-STAT1 → miR-29 → miR-29 target genes and its implication for melanoma growth.ResultsHere we show that IFN-γ induces STAT1-dependently a profound up-regulation of the miR-29 primary cluster pri-29a~b-1 in melanoma cell lines. Furthermore, expression levels of pri-29a~b-1 and mature miR-29a and miR-29b were elevated while the pri-29b-2~c cluster was almost undetectable. We observed an inverse correlation between miR-29a/b expression and the proliferation rate of various melanoma cell lines. This finding could be corroborated in cells transfected with either miR-29 mimics or inhibitors. The IFN-γ-induced G1-arrest of melanoma cells involves down-regulation of CDK6, which we proved to be a direct target of miR-29 in these cells. Compared to nevi and normal skin, and metastatic melanoma samples, miR-29a and miR-29b levels were found strikingly elevated in certain patient samples derived from primary melanoma.ConclusionsOur findings reveal that the miR-29a/b1 cluster is to be included in the group of IFN- and STAT-regulated genes. The up-regulated miR-29 family members may act as effectors of cytokine signalling in melanoma and other cancer cells as well as in the immune system.

MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs). To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs. Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines. We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients. Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p). Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.