A previously unknown Argonaute 2 variant positively modulates the viability of melanoma cells.

In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.

Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR.

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

MAGOH and MAGOHB Knockdown in Melanoma Cells Decreases Nonsense-Mediated Decay Activity and Promotes Apoptosis via Upregulation of GADD45A.

Cutaneous malignant melanoma is a highly proliferative and aggressive skin cancer with a steadily increasing incidence and a low long-term survival rate after metastatic progression. The protein MAGOH and its highly identical homologue MAGOHB are core components of the exon junction complex (EJC), which regulates splicing, stability and translation of mRNAs. The EJC, and especially MAGOH, has been shown to be involved in the development and progression of several cancers. In melanoma, the expression and function of both homologues remain essentially unexplored. This study identifies high MAGOH and MAGOHB protein expression in cutaneous melanoma cell lines and patient derived tissue samples. An siRNA-mediated knockdown of MAGOH significantly inhibits melanoma cell proliferation. The loss of MAGOH does not affect cell cycle progression, but induces apoptosis, an effect that is enhanced by a simultaneous knockdown of MAGOH and MAGOHB. MAGOH and MAGOHB do not influence the expression of the pro-apoptotic protein Bcl-XS or exon skipping. However, the knockdown of MAGOH and MAGOHB strongly decreases nonsense-mediated decay (NMD) activity, leading to an upregulation of the pro-apoptotic protein GADD45A. In conclusion, simultaneous inhibition of MAGOH and MAGOHB expression substantially affects cell survival, indicating both MAGOH homologues as promising new targets for the treatment of melanoma.

Learning from Embryogenesis-A Comparative Expression Analysis in Melanoblast Differentiation and Tumorigenesis Reveals miRNAs Driving Melanoma Development.

Malignant melanoma is one of the most dangerous tumor types due to its high metastasis rates and a steadily increasing incidence. During tumorigenesis, the molecular processes of embryonic development, exemplified by epithelial-mesenchymal transition (EMT), are often reactivated. For melanoma development, the exact molecular differences between melanoblasts, melanocytes, and melanoma cells are not completely understood. In this study, we aimed to identify microRNAs (miRNAs) that promote melanoma tumorigenesis and progression, based on an in vitro model of normal human epidermal melanocyte (NHEM) de-differentiation into melanoblast-like cells (MBrCs). Using miRNA-sequencing and differential expression analysis, we demonstrated in this study that a majority of miRNAs have an almost equal expression level in NHEMs and MBrCs but are significantly differentially regulated in primary tumor- and metastasis-derived melanoma cell lines. Further, a target gene analysis of strongly regulated but functionally unknown miRNAs yielded the implication of those miRNAs in many important cellular pathways driving malignancy. We hypothesize that many of the miRNAs discovered in our study are key drivers of melanoma development as they account for the tumorigenic potential that differentiates melanoma cells from proliferating or migrating embryonic cells.

Dissimilar Appearances Are Deceptive-Common microRNAs and Therapeutic Strategies in Liver Cancer and Melanoma.

: In this review, we summarize the current knowledge on miRNAs as therapeutic targets in two cancer types that were frequently described to be driven by miRNAs-melanoma and hepatocellular carcinoma (HCC). By focusing on common microRNAs and associated pathways in these-at first sight-dissimilar cancer types, we aim at revealing similar molecular mechanisms that are evolved in microRNA-biology to drive cancer progression. Thereby, we also want to outlay potential novel therapeutic strategies. After providing a brief introduction to general miRNA biology and basic information about HCC and melanoma, this review depicts prominent examples of potent oncomiRs and tumor-suppressor miRNAs, which have been proven to drive diverse cancer types including melanoma and HCC. To develop and apply miRNA-based therapeutics for cancer treatment in the future, it is essential to understand how miRNA dysregulation evolves during malignant transformation. Therefore, we highlight important aspects such as genetic alterations, miRNA editing and transcriptional regulation based on concrete examples. Furthermore, we expand our illustration by focusing on miRNA-associated proteins as well as other regulators of miRNAs which could also provide therapeutic targets. Finally, design and delivery strategies of miRNA-associated therapeutic agents as well as potential drawbacks are discussed to address the question of how miRNAs might contribute to cancer therapy in the future.