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Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma
Published in
2010
PMID: 20461151
Volume: 16
   
Pages: 828 - 842
Abstract
Purpose: Previously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study, we studied the genes regulated by Ep-CAM in a retinoblastoma Y79 cell line in vitro using a combination of short interference RNA and microarray technology. Methods: Flow cytometry, quantitative reverse transcriptase PCR (Q-RT-PCR), and immunohistochemistry were performed to confirm the Ep-CAM re-expression in the Y79 cells treated with 5′-azacytidine (AZC). Ep-CAM expression in AZC-treated Y79 cells was silenced using synthetic anti-Ep-CAM short interference RNA, and whole genome microarray was performed to determine the gene expression changes post Ep-CAM knockdown. Ep-CAM inhibition was confirmed by Q-RT-PCR, western blotting, and immunofluorescence. Results: Ep-CAM expression was significantly restored in Y79 cells on day 5 of AZC treatment. Ep-CAM inhibition significantly affected Y79 cell proliferation. We identified 465 upregulated genes (≥1.0 fold) and 205 downregulated genes (≤0.5 fold) in response to knockdown of Ep-CAM. These genes regulate several aspects of tumor function, including cell survival/proliferation, DNA replication/transcription, apoptosis, and angiogenesis. Quantitative pathway analysis using Biointerpreter further revealed that the most pronounced effect of Ep-CAM knockdown was deregulation of pathways that include mitogen-activated protein kinase (MAP) kinase and tumor protein 53 (P53) pathways. Real-time Q-RT-PCR confirmed microarray gene expression changes for selected genes. Conclusions: Ep-CAM silencing significantly decreases Y79 cell proliferation and revealed a wide network of deregulated pathways in vitro. Future studies targeting Ep-CAM gene expression in vivo will help to delineate the mechanisms associated with Ep-CAM gene function in neoplastic transformation and define the potential for Ep-CAM-based molecular intervention in retinoblastoma patients. © 2010 Molecular Vision.
About the journal
JournalMolecular Vision
ISSN10900535
Open AccessNo
Concepts (41)
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    AZACITIDINE
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    EPITHELIAL CELL ADHESION MOLECULE
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    Mitogen activated protein kinase
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    Protein p53
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    Small interfering rna
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    Angiogenesis
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    Apoptosis
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    Article
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    Cell proliferation
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    Cell survival
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    Controlled study
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    Dna microarray
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    Dna replication
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    DNA TRANSCRIPTION
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    Flow cytometry
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    Gene expression regulation
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    Gene silencing
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    Genome analysis
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    Immunofluorescence test
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    Immunohistochemistry
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    In vitro study
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    Nucleotide sequence
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    Priority journal
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    Retinoblastoma
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    Reverse transcription polymerase chain reaction
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    Rna interference
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    UNINDEXED SEQUENCE
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    Upregulation
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    Western blotting
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    ANTIGENS, NEOPLASM
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    AZACITIDINE
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    CELL ADHESION MOLECULES
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    Cell line
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    Down-regulation
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    GENOME, HUMAN
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    Humans
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    Microarray analysis
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    MITOGEN-ACTIVATED PROTEIN KINASE KINASES
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    Reverse transcriptase polymerase chain reaction
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    Tumor suppressor protein p53
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    UP-REGULATION