Oncogenic and tumor suppressing miRNAs have emerged as key regulators of gene expression in many types of cancer including melanoma.,We utilized quantitative in situ hybridization (qISH) to evaluate the tumor suppressing properties of miRNA, miR-205 in a population of human tumors.,We hypothesize decreased miR-205 would be associated with more aggressive tumors.,Multiplexing miR-205 qISH with immunofluorescent assessment of S100/GP100 allowed us to quantitatively evaluate miR-205 expression using the AQUA method of quantitative immunofluorescence.,The specificity of the assay was validated using blocking oligos and transfected cell lines as controls.,Outcomes were assessed on the Yale Melanoma Discovery Cohort consisting of 105 primary melanoma specimens and validated on an independent set of 206 primary melanomas (Yale Melanoma Validation Cohort).,Measurement of melanoma cell miR-205 levels shows a significantly shorter melanoma specific survival in patients with low expression.,Multivariate analysis shows miR-205 levels are significantly independent of stage, age, gender and Breslow depth.,Low levels of melanoma cell miR-205 expression as quantified by ISH show worse outcome, supporting the role of miR-205 as a tumor suppressor miRNA.,The quantification of miR205 in situ suggests potential for the use of miRNAs in future prognostic or predictive models.
Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ∼50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized.,Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAFV600E protein.,To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAFV600E expression.,To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAFV600E-mutant human melanomas.,BRAFV600E regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts.,These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription.,Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs.,BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration.,BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11.,Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.