Biochemical and Biophysical Research Communications
Loss of annexin A1 expression in human breast cancer detected by multiple high-throughput analyses☆
Section snippets
Materials and methods
Sample collection and RNA extraction. Human breast tissues were collected according to NIH guidelines, and by protocols approved by the UCLA Institutional Review Board. Fresh surgical samples of breast cancer and normal breast tissues were grossly dissected to remove as much fat tissues as possible and then stored in liquid nitrogen. TriZol (Invitrogen, Carlsbad, CA) was used for total RNA extraction according to the manufacturer’s instructions. RNA quality was examined by
Differential gene expression detected by multiple HTA
CGAP employed several approaches, including EST and SAGE, to catalog all genes expressed during cancer development [3]. Through May 2004, the CGAP database has collected about four million EST and 12 million SAGE tags. To explore the feasibility of using this resource for measuring gene expression levels, Virtual Northern was used to study the expression of a panel of 30 known breast cancer-related genes in cancerous and benign breast tissues (Table 2). Microarray, SAGE, and EST vNorthern each
Multiple HTA
HTA has proven to be a powerful tool in novel gene discovery, classification, and prognosis prediction in cancers. Recent studies focused on identification of disease-related genes [17], [18], expressional profiling to classify cancer into various subtypes [19], [20], [21], and to predict prognosis [22], [23], [24]. Since different HTA have different sensitivities and/or specificities in target gene discovery, we hypothesized that a properly combined use of these technologies might improve the
Conclusion
In summary, four differentially expressed genes in human breast cancer, ERBB2, GATA3, AGR2, and ANXA1, were detected by multiple HTA consisting of microarray and bioinformatic SAGE and EST. The loss of ANXA1 was further confirmed both at the mRNA level by real-time RT-PCR and human breast cancer profiling array, and at the protein level by immunohistochemical staining. Our study suggests that multiple HTA is a useful strategy for discovering biomarkers with clinical relevance in cancer
Acknowledgments
We thank the UCLA DNA microarray core for technical assistance in microarray analysis and Rebecca Radbod for statistical analysis. This study is supported in part by grants from the National Cancer Institute (5R01 CA093736), the Gonda Foundation, the UCLA Human Gene Medicine Program, and the Early Detection Research Network (NCI CA986366).
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Abbreviations: ANXA1, annexin A1; BCRG, breast cancer-related gene; CGAP, cancer genome anatomy project; DCIS, ductal carcinoma in situ; EST, expressed sequence tag; HTA, high-throughput analysis; IDC, invasive ductal carcinoma; RT-PCR, reverse transcriptase polymerase chain reaction; SAGE, series analysis of gene expression; vNorthern, virtual Northern.