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 (>or=1.0 fold) and 205 downregulated genes (<or=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.