Cell type and culture condition-dependent alternative splicing in human breast cancer cells

We used splicing-sensitive microarrays to detect differences in alternative splicing between two breast cancer cell lines MCF7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative), as well as cultured human mammary epithelial cells (HMEC). Several splicing alterations in genes, including CD44, FAS, RBM9, HnRNPA/B, APLP2, and MYL6, were detected by the microarray, and verified by RT-PCR. We also compared splicing in these breast cancer cells cultured in either two-dimensional flat dishes (2-D) or in three-dimensional Matrigel (3-D) conditions. Only a subset of the splicing differences that distinguish MCF7 cells from MDA-MB-231 cells under 2-D culture condition is retained under 3-D conditions, suggesting that alternative splicing events are influenced by the geometry of the culture conditions of these cells. Keywords: Splicing-sensitive microarray The design of oligonucleotides is for the general detection of splicing variants from a given gene. The junction oligonucleotides consist of exon?exon junction sequence, because different splice variants will have different exon?exon junctions. The microarray also contains oligonucleotides that are complementary to flanking constitutive exons, and the alternative exon. This splicing-sensitive microarray was used because a signal derived from the hybridization to the constitutive exon oligonucleotides would, in theory, reflect the total amount of RNA from the particular gene, whereas hybridization signals from an exon-exon junction oligonucleotide would reflect the amount of RNA containing that particular junction. The ratio of hybridization intensity from an oligonucleotide spanning a specific exon-exon junction to that from a constitutive exon oligonucleotide would provide information about the level of that alternatively spliced RNA in the two comparison samples. Using this type of splicingsensitive array, one can acquire two sets of information from one array: expression level changes for the gene and changes in the distribution of the splice variants. The array used in this study contained 64 genes that underwent alternative splicing, including estrogen receptor 1 and 2 (ER1 and ER2), CD44 (cell adhesion molecule), ITGA6 (integrin a6 precursor), FAS, LARD, WT1 (Wilm?s tumor protein 1), and TP73 (tumor suppressor p73). Each gene could contain one or more simple alternative exons, or more complex arrangements of multiple alternative exons.