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A Functional Screen Identifies Specific MicroRNAs Capable of Inhibiting Human Melanoma Cell Viability

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      Abstract

      Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

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      Most cited references 45

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      MicroRNAs: genomics, biogenesis, mechanism, and function.

       David Bartel (2004)
      MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
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        Mutations of the BRAF gene in human cancer.

        Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
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          Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.

          MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.
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            Author and article information

            Affiliations
            [1 ]Hubrecht Institute, University Medical Center Utrecht, Cancer Genomics Center, Utrecht, The Netherlands
            [2 ]InteRNA Technologies BV, Utrecht, The Netherlands
            University of Connecticut Health Center, United States of America
            Author notes

            Competing Interests: Dr. van Haastert, Dr. Gommans, Dr. van Noort, Dr. Schaapveld are employees of InteRNA Technologies and hold shares in the company. InteRNA Technologies has filed for a patent on therapeutic application for cancer treatment using specific miRNAs as described in this manuscript. This application is currently in the PTC phase. The claims in this patent do not alter the authors' adherence to or interfere with all the PLoS ONE policies on sharing data and materials.

            Conceived and designed the experiments: JP RH WG PN GP RS EC. Performed the experiments: JP RH TG IS WG MV FC. Analyzed the data: JP RH FC. Wrote the paper: JP RH EC.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2012
            22 August 2012
            : 7
            : 8
            3425484
            22927992
            PONE-D-11-23134
            10.1371/journal.pone.0043569
            (Editor)

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Pages: 11
            Funding
            This work was financially supported by the Cancer Genomics Center through grants from the Netherlands Genomics Initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology
            Molecular Cell Biology
            Nucleic Acids
            RNA
            RNA interference
            Medicine
            Dermatology
            Skin Neoplasms
            Malignant Skin Neoplasms
            Melanomas
            Oncology
            Cancers and Neoplasms
            Skin Tumors
            Malignant Melanoma
            Basic Cancer Research
            Cancer Treatment

            Uncategorized

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