Related Experiment Videos
Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma
Moutushy Mitra1, Mallikarjuna Kandalam, Rama Shanker Verma
1Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
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 (
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.
Insights
Epithelial cell adhesion molecule (Ep-CAM) silencing in retinoblastoma Y79 cells reduced proliferation and altered gene expression. This study identifies key pathways affected by Ep-CAM knockdown, offering potential for targeted therapies.
Area of Science:
- Oncology
- Molecular Biology
- Ophthalmology
Background:
- Epithelial cell adhesion molecule (Ep-CAM) is highly expressed in retinoblastoma.
- Understanding Ep-CAM's regulatory role is crucial for retinoblastoma treatment.
Purpose of the Study:
- To investigate genes regulated by Ep-CAM in retinoblastoma Y79 cells.
- To explore the impact of Ep-CAM knockdown on retinoblastoma cell function.
Main Methods:
- Ep-CAM re-expression confirmed using flow cytometry, Q-RT-PCR, and immunohistochemistry.
- Ep-CAM knockdown achieved via short interference RNA in Y79 cells.
- Whole genome microarray analysis identified gene expression changes post-Ep-CAM inhibition.
Main Results:
- Ep-CAM inhibition significantly decreased Y79 cell proliferation.
- Identified 465 upregulated and 205 downregulated genes upon Ep-CAM knockdown.
- Deregulation of MAPK and p53 pathways observed, impacting cell survival, proliferation, and angiogenesis.
Conclusions:
- Ep-CAM silencing reduces retinoblastoma cell proliferation and impacts multiple signaling pathways.
- Further in vivo studies are needed to explore Ep-CAM's role in neoplastic transformation.
- Ep-CAM may represent a potential therapeutic target for retinoblastoma.
Related Concept Videos
01:20The Retinoblastoma Gene
The first-ever tumor suppressor gene called Rb was identified in retinoblastoma - a rare eye tumor in children. In inherited forms of the disease, a child inherits one defective copy of the Rb gene, which predisposes them to retinoblastoma. However,...
01:20The Retinoblastoma Gene
The first-ever tumor suppressor gene called Rb was identified in retinoblastoma - a rare eye tumor in children. In inherited forms of the disease, a child inherits one defective copy of the Rb gene, which predisposes them to retinoblastoma. However,...