title: Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts.
abstract: 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.
authors.affiliation: Laboratory of Genomics and Molecular Biology, Hospital A.C. Camargo, Fundação Antonio Prudente, São Paulo, 01509-900, Brazil.
authors.forename: Elisa N
authors.initials: EN
authors.lastname: Ferreira
authors.forename: Maria C R
authors.initials: MC
authors.lastname: Rangel
authors.forename: Pedro F
authors.initials: PF
authors.lastname: Galante
authors.forename: Jorge E
authors.initials: JE
authors.lastname: de Souza
authors.forename: Gustavo C
authors.initials: GC
authors.lastname: Molina
authors.forename: Sandro J
authors.initials: SJ
authors.lastname: de Souza
authors.forename: Dirce M
authors.initials: DM
authors.lastname: Carraro
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019295:Computational Biology; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D009841:Oligonucleotides; D018719:Erb-b2 Receptor Tyrosine Kinases