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Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts
BACKGROUND: Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large sp...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045797/ https://www.ncbi.nlm.nih.gov/pubmed/21210970 http://dx.doi.org/10.1186/1471-2164-11-S5-S4 |
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author | Ferreira, Elisa N Rangel, Maria CR Galante, Pedro F de Souza, Jorge E Molina, Gustavo C de Souza, Sandro J Carraro, Dirce M |
author_facet | Ferreira, Elisa N Rangel, Maria CR Galante, Pedro F de Souza, Jorge E Molina, Gustavo C de Souza, Sandro J Carraro, Dirce M |
author_sort | Ferreira, Elisa N |
collection | PubMed |
description | BACKGROUND: Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. RESULTS: The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. CONCLUSIONS: In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers. |
format | Text |
id | pubmed-3045797 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30457972011-03-01 Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts Ferreira, Elisa N Rangel, Maria CR Galante, Pedro F de Souza, Jorge E Molina, Gustavo C de Souza, Sandro J Carraro, Dirce M BMC Genomics Proceedings BACKGROUND: Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. RESULTS: The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. CONCLUSIONS: In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers. BioMed Central 2010-12-22 /pmc/articles/PMC3045797/ /pubmed/21210970 http://dx.doi.org/10.1186/1471-2164-11-S5-S4 Text en Copyright ©2010 Carraro et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Proceedings Ferreira, Elisa N Rangel, Maria CR Galante, Pedro F de Souza, Jorge E Molina, Gustavo C de Souza, Sandro J Carraro, Dirce M Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts |
title | Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts |
title_full | Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts |
title_fullStr | Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts |
title_full_unstemmed | Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts |
title_short | Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts |
title_sort | alternative splicing enriched cdna libraries identify breast cancer-associated transcripts |
topic | Proceedings |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045797/ https://www.ncbi.nlm.nih.gov/pubmed/21210970 http://dx.doi.org/10.1186/1471-2164-11-S5-S4 |
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