title: A dedicated microarray for in-depth analysis of pre-mRNA splicing events: application to the study of genes involved in the response to targeted anticancer therapies.
abstract: Alternative pre-mRNA splicing (AS) widely expands proteome diversity through the combinatorial assembly of exons. The analysis of AS on a large scale, by using splice-sensitive microarrays, is a highly efficient method to detect the majority of known and predicted alternative transcripts for a given gene. The response to targeted anticancer therapies cannot easily be anticipated without prior knowledge of the expression, by the tumor, of target proteins or genes. To analyze, in depth, transcript structure and levels for genes involved in these responses, including AKT1-3, HER1-4, HIF1A, PIK3CA, PIK3R1-2, VEGFA-D and PIR, we engineered a dedicated gene chip with coverage of an average 185 probes per gene and, especially, exon-exon junction probes. As a proof of concept, we demonstrated the ability of such a chip to detect the effects of over-expressed SRSF2 RNA binding protein on the structure and abundance of mRNA products in H358 lung cancer cells conditionally over-expressing SRSF2. Major splicing changes were observed, including in HER1/EGFR pre-mRNA, which were also seen in human lung cancer samples over-expressing the SRSF2 protein. In addition, we showed that variations in HER1/EGFR pre-mRNA splicing triggered by SRSF2 overexpression in H358 cells resulted in a drop in HER1/EGFR protein level, which correlated with increased sensitivity to gefitinib, an EGFR tyrosine kinase inhibitor. We propose, therefore, that this novel tool could be especially relevant for clinical applications, with the aim to predict the response before treatment.
authors.forename: Marine
authors.initials: M
authors.lastname: Pesson
authors.forename: Béatrice
authors.initials: B
authors.lastname: Eymin
authors.forename: Pierre
authors.initials: P
authors.lastname: De La Grange
authors.forename: Brigitte
authors.initials: B
authors.lastname: Simon
authors.affiliation: UMR INSERM U1078-UBO, Equipe ECLA, Faculté de Médecine, 22 Avenue Camille Desmoulins, 29200 Brest, France. laurent.corcos@inserm.fr.
authors.forename: Laurent
authors.initials: L
authors.lastname: Corcos
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics; D017398:Alternative Splicing; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D058990:Molecular Targeted Therapy; D009687:Nuclear Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012322:RNA Precursors / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000235:genetics; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors