Genomic and functional characterization of RMEL3, a long noncoding RNA with melanoma-enriched expression

Author(s)

Lucas Goedert

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-08-02

Examining board members

Enilza Maria Espreafico; Leticia Fröhlich Archangelo; Patricia Abrão Possik

• Advisor: Enilza Maria Espreafico

Abstract

Previous work by our group identified RMEL3 as a long non-coding RNA (lncRNA) with melanoma enriched expression. In the present work, through the analyses of the melanoma public data of The Cancer Genome Atlas (TCGA), we confirmed RMEL3 enriched expression in melanoma compared to normal tissues and other types of cancers. We identified that melanoma patients bearing the BRAFV600E mutation have a higher RMEL3 expression and have shown that the blockage of this oncoprotein reduces the expression levels of RMEL3 by 60%, revealing the activated BRAF as an upstream regulator of this lncRNA. RMEL3 shRNA-mediated silencing drastically reduced colony formation (90%) and migration (85%) in the A375 melanoma cell line, which is supported by data showing higher RMEL3 expression in phenotypically invasive tumors and in tumors at the onset of metastasis. The observed phenotype is, at least partially, explained by the transcriptomic profile associated with this lncRNA, which includes alterations in p53 signaling and epithelial-mesenchymal transition, as well as a strong correlation with components and effectors of the MAPK and PI3K pathways. We identified the existence of different RMEL3 isoforms with distinct intracellular distributions, which support their performance in independent molecular functions. The analysis of the TCGA database revealed the possibility of RMEL3 acting as a sponge for microRNAs favoring the expression of protumorigenic genes like FGF2 and Bcl-2 and, in addition, the expression of RMEL3 was shown to be strongly correlated with a genome hypermethylation and appears to counterbalance BRAFV600E-induced hypomethylation, affecting different targets. Futhermore, the pulldown assay of the RMEL3 native transcript followed by mass spectrometry identified a number of RMEL3 binding proteins involved in processes that are known to support tumor progression, such as glycolysis and the MAPK pathway. Thus, the TCGA patient data set and functional data demonstrate that RMEL3 is important for MAPK and PI3K signaling and its silencing negatively affects aspects that support the malignancy of melanomas harboring the BRAFV600E mutation. (AU)

• FAPESP's process: 14/07726-0 - Genomic and functional characterization of genes with expression restricted to melanoma (RMELs)
• Grantee: Lucas Goedert
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)