microRNAs contribute to melanoma development
The best-studied group of non-coding RNAs are miRNAs, which in most cases decrease targeted mRNA levels . For a general review of miRNAs in cancer, see reference . miRNAs can act as tumor suppressors as well as oncogenes , and there is no doubt about their role in tumorigenesis . In tumors, the biogenesis of miRNAs is disturbed [32, 33], which will alter the expression levels of miRNAs and ultimately the expression of genes regulated by miRNAs . In melanoma, the miRNAome  has been determined, and the role of miRNAs in melanomas has been reviewed [35-37]. miRNAs are involved in all steps of tumorigenesis from initiation  to metastasis [39, 40]. Melanoma subtypes differ in their miRNA signatures , which can serve as a prognostic biomarker . Additionally, miRNAs regulate not only mRNAs but also other ncRNAs [43, 44], and they themselves are epigenetically regulated . This places ncRNAs in a wider, multilayer regulatory network of transcriptional and translational control (Fig. 2).
Figure 2. Simplified map of interactions in the non-coding RNA network itself and with mRNA expression. The symbols mean ⊥ inhibition, stimulation, ⟷ mutual interaction.
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Do lncRNAs have a role in melanomagenesis?
Besides miRNAs, there are also lncRNAs . As implied by their name, they are larger than miRNAs with a minimum length of 200 bp and up to several kilobases. One of the best-studied members is XIST, which is involved in the inactivation of the X chromosome . lncRNAs are divided in three major groups: long intergenic non-coding RNAs (lincRNA), which are located away from protein-coding regions, antisense RNAs (asRNA), which are transcribed in reverse orientation and overlap with a known gene, and intronic lncRNAs. The groups have partly overlapping activities. For example, members of each group regulate epigenetic modifications; however, only the transcription of asRNAs directly interferes with transcription of its corresponding sense counterpart by polymerase stalling. The different functions attributed to lncRNAs [reviewed in [48, 49]] include control of pluripotency and differentiation in stem cells , setting of epigenetic marks  and functioning as enhancer RNAs . One subgroup of intron-derived lncRNAs, the sno-lncRNAs, is associated with changes in splicing . Although lncRNAs are expressed in normal cells with tissue-specific expression patterns, aberrant expression occurs in tumors  and they play roles in cancer progression . Based on the expanding knowledge of their regulatory influence, lncRNAs may represent ‘a new frontier of translational research’ in cancer . There is very limited information on the role of specific lncRNAs in melanoma. In 2011, Khaitan et al.  showed that the lncRNA SPRY4-IT1 modulates cell growth and differentiation and its knock-down increased apoptosis. Flockhart et al.  described the effect of V600EBRAF on the expression of about 100 lncRNAs.
The number of known lncRNAs is still increasing. The subclass lincRNAs alone contains more than 8000 members  with different activities. One of the described functions of lncRNAs is setting of epigenetic marks. For example, the lncRNA HOTAIR is involved in the setting of epigenetic marks by the polycomb repressive complex 2, and its expression levels are increased in breast tumors. A high expression level implies a poor prognosis for metastasis and survival in breast tumor patients . It is well established that epigenetic changes occur in tumors [61, 62] and that these changes play a role in melanomas as well . Interestingly, conditions that are indirectly linked to the onset of tumors, such as stress [64, 65] and age , induce epigenetic changes. Issa and Garber suggested the presence of an epigenetic predisposition to cancer . Therefore, epigenetic changes induced by dysregulation of ncRNAs may act like an oncogenic event. One has to emphasize that different non-coding RNAs, lincRNAs, asRNAs  as well as miRNAs can induce epigenetic changes, but at the same time, miRNAs are regulated by epigenetic changes .
The next group of lncRNAs are asRNAs, which contain not only non-coding RNAs but also coding RNAs although to a lesser extent (e.g. FGF-2 and its asRNA FGF-AS ). AsRNAs are relatively common [70, 71]. They regulate the expression of their corresponding sense genes by different mechanisms , like influencing sense RNA stability, epigenetic changes and alternative splicing [73, 74]. The idea that splicing is a factor in tumorigenesis is not new [75, 76], but the study and detection of splice variants were previously hampered by technical limitations. With increasing numbers of high-throughput RNA sequencing studies completed (reviewed in ), we will obtain a more complete list of splice variations  between normal and diseased tissues and thus a better understanding of their role in cancer, including melanoma. In mice, melanocytes and melanomas differ in splice variants . Even more astonishing is the observation that breast cancer cells grown in 2D or 3D culture differ in their splice variants , which suggests that alternative splicing can occur in response to subtle changes in the microenvironment. In melanoma, different splice isoforms of MITF, the master regulator of melanocyte development, have been described  as well as aberrant splicing of the tumor suppressor Bin1 . Splicing even plays a role in resistance to cancer treatments such as vemurafenib . Based on these findings, targeting specific splice variants by antisense oligonucleotides has been suggested for cancer therapy , and splice variants have been proposed as cancer biomarkers .
Alternative splicing is not an isolated incident. Splice variants of DNA methyltransferases  differ in their activity, which may result in epigenetic changes. At the same time, epigenetic changes regulate alternative splicing . Additionally, changes in the 3′UTR of mRNAs due to alternative splicing can alter the recognition by miRNAs . Thus, alternative splicing is part of a larger interacting network and is influenced by ncRNAs .
Are non-coding RNAs the new frontier in melanoma research?
Biological and bioinformatics analyses of non-coding regions are still in their infancy, with the exception of the well-established miRNA field. The cancer genome atlas is just the beginning, and much research remains to be carried out. We will gather much information on exon sequences, genomic deletions and amplifications, but like first graders, we still lack the knowledge to fully comprehend the information contained in the whole genomic sequence. We still do not know how to interpret the sequences and possible mutations of non-coding RNAs. In contrast to protein-coding sequences, no reference sequences exist for those regions to define wild-type versus mutated versions. Because the functions of most ncRNAs are unknown, no assays are available to determine which sequence changes influence their function.
Analysing each single compartment (protein, mRNA, ncRNA) and their interactions is already a major challenge. However, the analyses of the key players are further hindered by the fact that splice variants, epigenetic changes and ncRNAs interact and influence each other. For example, histone modifications regulate alternative splicing , and conversely, splice variants of DNA methyltransferases result in epigenetic changes . All these data stress the fact that control of mRNA expression (transcription factors, translation, etc.) is overlaid and controlled by an interacting network of ncRNAs (Fig. 2). Any disturbance at any level, including ncRNA interactions, will eventually have consequences at the protein level. This will either be reflected in the quantity of protein or in introduced modifications (e.g. alternative splicing).