Transcription profiling of human MCF10A cells in 3-D culture for 15 h after treatment with CtIP-RNAi vs Luc-RNAi adenovirus

BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen of genes including angiopoietin-1 (ANG1), a secreted angiogenic factor, are co-repressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3-D culture. BRCA1, CtIP and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells forming capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization and overexpressed ANG1. These results suggest, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment. Experiment Overall Design: MCF10A cells seeded at 5x105 cells/60mm plate were infected with adenoviral luciferase- or CtIP-RNAi in duplicate at 20 MOI for 24h. Infected cells were re-seeded at 5x105 cells in a 60mm plate pre-coated with Growth Factor Reduced Matrigel and covered with the growth medium containing 2% Matrigel at 37°C for 15h. RNA was extracted and quality assessed. cDNA synthesized from the harvested RNA was biotin-labeled, hybridized onto Affymetrix HG U133 PLUS 2.0 array (54,676 genes) and stained with streptavidin-phycoerythrin. The hybridized array was analyzed using GeneChip® Scanner 3000 and GCOS 1.2 software (Affymetrix) at the UCI Microarray Core service.