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Novel primate-specific genes, RMEL 1, 2 and 3, with highly restricted expression in melanoma, assessed by new data mining tool.

Sousa JF, Torrieri R, Silva RR, Pereira CG, Valente V, Torrieri E, Peronni KC, Martins W, Muto N, Francisco G, Brohem CA, Carlotti CG, Maria-Engler SS, Chammas R, Espreafico EM - PLoS ONE (2010)

Bottom Line: We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression.Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes.They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

ABSTRACT
Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

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Validation by real-time RT-PCR of the melanoma/melanocyte-restricted expression of RMEL1, 2 and 3.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the sample displaying the lowest normalized Ct as reference. The tissue panel included normal melanocytes (NM), melanoma cell lines (melanoma), including those harboring the activating BRAF V600E mutation (*), wild type BRAF (not marked), or with mutation status not determined (ND); and various cell lines and tissues of non-melanocytic origin (non-melanoma), including primary skin fibroblasts, HeLa, HL-60, Jurkat cells, peripheral blood mononuclear cells (PBMC), and necropsy samples from spleen, brain, esophagus, liver, intestine, skeletal muscle, kidney, normal bladder (NB), bladder tumor (BT), normal prostate (NP), prostate tumor (PT), normal glia (NG), gliobastoma (GBM).
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pone-0013510-g001: Validation by real-time RT-PCR of the melanoma/melanocyte-restricted expression of RMEL1, 2 and 3.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the sample displaying the lowest normalized Ct as reference. The tissue panel included normal melanocytes (NM), melanoma cell lines (melanoma), including those harboring the activating BRAF V600E mutation (*), wild type BRAF (not marked), or with mutation status not determined (ND); and various cell lines and tissues of non-melanocytic origin (non-melanoma), including primary skin fibroblasts, HeLa, HL-60, Jurkat cells, peripheral blood mononuclear cells (PBMC), and necropsy samples from spleen, brain, esophagus, liver, intestine, skeletal muscle, kidney, normal bladder (NB), bladder tumor (BT), normal prostate (NP), prostate tumor (PT), normal glia (NG), gliobastoma (GBM).

Mentions: For validation experiments we selected UniGene clusters composed of three or more GenBank sequences (Tables S2 and S4), excluding Hs.551051 and Hs.617329 that required further analysis to clarify whether they represent a single gene. Primer efficiency tests revealed six of nine genes with very low expression levels (Ct>34). Thus, we focused on the other three genes (Hs.295012, Hs.518390, Hs.559350) showing more consistent and reliable expression levels. As shown in Figure 1, expression analysis in a diverse panel of human cell lines and tissues confirmed highly restricted expression of these three genes in melanocyte/melanoma cells, so we named them as RMEL1 (Hs.295012), RMEL2 (Hs.518391), and RMEL3 (Hs.559350). Except for very low levels found in a single glioblastoma sample (GBM-2), RMEL1 mRNA expression was detected only in samples from melanocytic origin, including the two primary melanocyte cultures and 14 of 19 melanoma cell lines, confirming its highly restricted expression in melanocytic cells (Figure 1A). RMEL2 mRNA expression was detected in 13 out of 19 melanoma cell lines, and no expression was detected in melanocytes, nor in 29 samples from other cell and tissue types (Figure 1B). However, despite the lack of expression in normal glia, high levels were detected in one (GMB-3) out of three glioblastoma samples (Figure 1B), suggesting that this gene, and perhaps RMEL1 as well, are part of a common tumorigenic tract involved in these two types of neuroectoderm-derived tumors. RMEL3 mRNA expression was detected in 13 out of 19 melanoma cell lines and in low levels in two normal bladder samples and one prostate tumor (Figure 1C). No expression was detected in melanocytes nor in 28 samples from other types of cells and tissues. Interestingly, there was a positive correlation between the expression of these three genes and the occurrence of oncogenic mutation of BRAF (Figure 1A–C).


Novel primate-specific genes, RMEL 1, 2 and 3, with highly restricted expression in melanoma, assessed by new data mining tool.

Sousa JF, Torrieri R, Silva RR, Pereira CG, Valente V, Torrieri E, Peronni KC, Martins W, Muto N, Francisco G, Brohem CA, Carlotti CG, Maria-Engler SS, Chammas R, Espreafico EM - PLoS ONE (2010)

Validation by real-time RT-PCR of the melanoma/melanocyte-restricted expression of RMEL1, 2 and 3.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the sample displaying the lowest normalized Ct as reference. The tissue panel included normal melanocytes (NM), melanoma cell lines (melanoma), including those harboring the activating BRAF V600E mutation (*), wild type BRAF (not marked), or with mutation status not determined (ND); and various cell lines and tissues of non-melanocytic origin (non-melanoma), including primary skin fibroblasts, HeLa, HL-60, Jurkat cells, peripheral blood mononuclear cells (PBMC), and necropsy samples from spleen, brain, esophagus, liver, intestine, skeletal muscle, kidney, normal bladder (NB), bladder tumor (BT), normal prostate (NP), prostate tumor (PT), normal glia (NG), gliobastoma (GBM).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958148&req=5

pone-0013510-g001: Validation by real-time RT-PCR of the melanoma/melanocyte-restricted expression of RMEL1, 2 and 3.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the sample displaying the lowest normalized Ct as reference. The tissue panel included normal melanocytes (NM), melanoma cell lines (melanoma), including those harboring the activating BRAF V600E mutation (*), wild type BRAF (not marked), or with mutation status not determined (ND); and various cell lines and tissues of non-melanocytic origin (non-melanoma), including primary skin fibroblasts, HeLa, HL-60, Jurkat cells, peripheral blood mononuclear cells (PBMC), and necropsy samples from spleen, brain, esophagus, liver, intestine, skeletal muscle, kidney, normal bladder (NB), bladder tumor (BT), normal prostate (NP), prostate tumor (PT), normal glia (NG), gliobastoma (GBM).
Mentions: For validation experiments we selected UniGene clusters composed of three or more GenBank sequences (Tables S2 and S4), excluding Hs.551051 and Hs.617329 that required further analysis to clarify whether they represent a single gene. Primer efficiency tests revealed six of nine genes with very low expression levels (Ct>34). Thus, we focused on the other three genes (Hs.295012, Hs.518390, Hs.559350) showing more consistent and reliable expression levels. As shown in Figure 1, expression analysis in a diverse panel of human cell lines and tissues confirmed highly restricted expression of these three genes in melanocyte/melanoma cells, so we named them as RMEL1 (Hs.295012), RMEL2 (Hs.518391), and RMEL3 (Hs.559350). Except for very low levels found in a single glioblastoma sample (GBM-2), RMEL1 mRNA expression was detected only in samples from melanocytic origin, including the two primary melanocyte cultures and 14 of 19 melanoma cell lines, confirming its highly restricted expression in melanocytic cells (Figure 1A). RMEL2 mRNA expression was detected in 13 out of 19 melanoma cell lines, and no expression was detected in melanocytes, nor in 29 samples from other cell and tissue types (Figure 1B). However, despite the lack of expression in normal glia, high levels were detected in one (GMB-3) out of three glioblastoma samples (Figure 1B), suggesting that this gene, and perhaps RMEL1 as well, are part of a common tumorigenic tract involved in these two types of neuroectoderm-derived tumors. RMEL3 mRNA expression was detected in 13 out of 19 melanoma cell lines and in low levels in two normal bladder samples and one prostate tumor (Figure 1C). No expression was detected in melanocytes nor in 28 samples from other types of cells and tissues. Interestingly, there was a positive correlation between the expression of these three genes and the occurrence of oncogenic mutation of BRAF (Figure 1A–C).

Bottom Line: We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression.Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes.They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

ABSTRACT
Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Show MeSH
Related in: MedlinePlus