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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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Expression profiles of RMEL1, 2 and 3 during melanoma progression.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the mean value of the normalized Cts of all nevi samples as reference. cDNA samples derived from keratynocytes (Ker), nevi (N), primary melanoma tumors (P) and melanoma metastasis (M), from different patients (distinguished with a number following the letter N, P or M) were analyzed by Real-Time PCR. Statistical analyses were performed using ANOVA (after loge transformation of the data), for RMEL1 and RMEL2, or the Mann–Whitney test for RMEL3. Metastatic samples numbered 79, 81 and 105 are from lymph node metastasis; 50, 80, 93, 95, 96, 108, 113 and 127 from skin metastasis; 106 from lung metastasis and 82, 83, 116 and 129 from visceral metastasis.
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pone-0013510-g002: Expression profiles of RMEL1, 2 and 3 during melanoma progression.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the mean value of the normalized Cts of all nevi samples as reference. cDNA samples derived from keratynocytes (Ker), nevi (N), primary melanoma tumors (P) and melanoma metastasis (M), from different patients (distinguished with a number following the letter N, P or M) were analyzed by Real-Time PCR. Statistical analyses were performed using ANOVA (after loge transformation of the data), for RMEL1 and RMEL2, or the Mann–Whitney test for RMEL3. Metastatic samples numbered 79, 81 and 105 are from lymph node metastasis; 50, 80, 93, 95, 96, 108, 113 and 127 from skin metastasis; 106 from lung metastasis and 82, 83, 116 and 129 from visceral metastasis.

Mentions: We next analyzed the expression profile of these genes in a panel of nevi, primary, and metastatic melanoma tissues, including a sample from primary cultured keratinocytes as control (Figure 2). Real-time RT-PCR results showed lack of mRNA expression in keratinocytes, while significant differential expression was detected for the three genes in nevi and melanomas of both stages. Both RMEL1 and RMEL2 showed up-regulation in about 45 to 50% of the tumor samples. On the other hand, RMEL3 exhibited the inverse expression pattern, marked by relatively high levels in nevi and progressive loss during melanoma progression, as revealed by its loss in 31% of the primary tumors and in 88% of the metastatic tumors (Figure 2A–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)

Expression profiles of RMEL1, 2 and 3 during melanoma progression.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the mean value of the normalized Cts of all nevi samples as reference. cDNA samples derived from keratynocytes (Ker), nevi (N), primary melanoma tumors (P) and melanoma metastasis (M), from different patients (distinguished with a number following the letter N, P or M) were analyzed by Real-Time PCR. Statistical analyses were performed using ANOVA (after loge transformation of the data), for RMEL1 and RMEL2, or the Mann–Whitney test for RMEL3. Metastatic samples numbered 79, 81 and 105 are from lymph node metastasis; 50, 80, 93, 95, 96, 108, 113 and 127 from skin metastasis; 106 from lung metastasis and 82, 83, 116 and 129 from visceral metastasis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958148&req=5

pone-0013510-g002: Expression profiles of RMEL1, 2 and 3 during melanoma progression.Relative expression was calculated according to 2−ΔΔCT method, using TBP (Tata-box binding protein) as endogenous control and the mean value of the normalized Cts of all nevi samples as reference. cDNA samples derived from keratynocytes (Ker), nevi (N), primary melanoma tumors (P) and melanoma metastasis (M), from different patients (distinguished with a number following the letter N, P or M) were analyzed by Real-Time PCR. Statistical analyses were performed using ANOVA (after loge transformation of the data), for RMEL1 and RMEL2, or the Mann–Whitney test for RMEL3. Metastatic samples numbered 79, 81 and 105 are from lymph node metastasis; 50, 80, 93, 95, 96, 108, 113 and 127 from skin metastasis; 106 from lung metastasis and 82, 83, 116 and 129 from visceral metastasis.
Mentions: We next analyzed the expression profile of these genes in a panel of nevi, primary, and metastatic melanoma tissues, including a sample from primary cultured keratinocytes as control (Figure 2). Real-time RT-PCR results showed lack of mRNA expression in keratinocytes, while significant differential expression was detected for the three genes in nevi and melanomas of both stages. Both RMEL1 and RMEL2 showed up-regulation in about 45 to 50% of the tumor samples. On the other hand, RMEL3 exhibited the inverse expression pattern, marked by relatively high levels in nevi and progressive loss during melanoma progression, as revealed by its loss in 31% of the primary tumors and in 88% of the metastatic tumors (Figure 2A–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