title: Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Moutushy
authors.initials: M
authors.lastname: Mitra
authors.forename: Mallikarjuna
authors.initials: M
authors.lastname: Kandalam
authors.forename: Rama Shanker
authors.initials: RS
authors.lastname: Verma
authors.forename: Krishnan
authors.initials: K
authors.lastname: UmaMaheswari
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnakumar
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001374:Azacitidine / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D015536:Down-Regulation; D000071858:Epithelial Cell Adhesion Molecule; D005786:Gene Expression Regulation; D020868:Gene Silencing; D015894:Genome, Human; D006801:Humans; D046228:Microarray Analysis; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D016159:Tumor Suppressor Protein p53; D020929:Mitogen-Activated Protein Kinase Kinases; D001374:Azacitidine
references.citation: J Pathol. 1999 Jun;188(2):201-6
references.pmid: 10398165
references.citation: Eur Urol. 1995;28(3):251-4
references.pmid: 8536781
references.citation: J Cell Biochem. 2005 Dec 1;96(5):906-13
references.pmid: 16163738
references.citation: Cancer Lett. 2007 Feb 8;246(1-2):253-61
references.pmid: 16624485
references.citation: J Mol Med (Berl). 1999 Oct;77(10):699-712
references.pmid: 10606205
references.citation: Clin Cancer Res. 2005 Jun 15;11(12):4321-30
references.pmid: 15958613
references.citation: Am J Pathol. 2003 Dec;163(6):2139-48
references.pmid: 14633587
references.citation: Cytogenet Cell Genet. 1975;14(1):9-25
references.pmid: 1093816
references.citation: Bone Marrow Transplant. 2003 Feb;31(4):281-4
references.pmid: 12621463
references.citation: Br J Cancer. 2007 Feb 12;96(3):417-23
references.pmid: 17211480
references.citation: Nucleic Acids Res. 2002 Jan 1;30(1):207-10
references.pmid: 11752295
references.citation: Blood. 1999 Jun 15;93(12):4059-70
references.pmid: 10361102
references.citation: Oncogene. 2004 Jul 29;23(34):5748-58
references.pmid: 15195135
references.citation: J Pediatr Hematol Oncol. 2007 Jun;29(6):399-405
references.pmid: 17551402
references.citation: Clin Cancer Res. 1999 Dec;5(12):3999-4004
references.pmid: 10632331
references.citation: J Clin Oncol. 1996 May;14(5):1532-6
references.pmid: 8622068
references.citation: Cancer. 1994 Jul 15;74(2):722-32
references.pmid: 8033054
references.citation: Nature. 2001 May 24;411(6836):494-8
references.pmid: 11373684
references.citation: Invest Ophthalmol Vis Sci. 2004 Dec;45(12):4247-50
references.pmid: 15557427
references.citation: Hematol Oncol Clin North Am. 1987 Dec;1(4):721-35
references.pmid: 3323180
references.citation: Cell. 2007 Feb 23;128(4):683-92
references.pmid: 17320506
references.citation: J Cancer Res Clin Oncol. 2009 Sep;135(9):1277-85
references.pmid: 19294417
references.citation: Genes Chromosomes Cancer. 2006 Jan;45(1):72-82
references.pmid: 16180235
references.citation: Somatic Cell Genet. 1979 Nov;5(6):957-71
references.pmid: 94699
references.citation: Cancer Res. 2004 Aug 15;64(16):5818-24
references.pmid: 15313925
references.citation: EMBO J. 2007 Feb 7;26(3):784-94
references.pmid: 17235288
references.citation: Int J Cancer. 2008 Jul 15;123(2):484-489
references.pmid: 18398839
references.citation: Cell Death Differ. 2005 May;12(5):453-62
references.pmid: 15933725
references.citation: Nat Cell Biol. 2009 Feb;11(2):162-71
references.pmid: 19136966
references.citation: J Pediatr Ophthalmol Strabismus. 2003 Sep-Oct;40(5):265-7
references.pmid: 14560832
references.citation: Cancer Lett. 2003 Apr 10;193(1):25-32
references.pmid: 12691820
references.citation: Exp Eye Res. 2006 Dec;83(6):1446-52
references.pmid: 17027002
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Am J Pathol. 2000 Oct;157(4):1405-12
references.pmid: 11021842
references.citation: Oncogene. 2007 Jun 7;26(27):3989-97
references.pmid: 17213811
references.citation: Int J Clin Oncol. 2004 Feb;9(1):1-6
references.pmid: 15162819
references.citation: Cancer Genet Cytogenet. 1988 Jan;30(1):119-26
references.pmid: 3422037
references.citation: Clin Cancer Res. 2004 Apr 15;10(8):2659-69
references.pmid: 15102668
references.citation: Cell. 2006 Jul 14;126(1):121-34
references.pmid: 16839881
references.citation: Oral Oncol. 2009 Jan;45(1):e1-8
references.pmid: 18485790
references.citation: Hum Mol Genet. 2008 May 15;17(10):1363-72
references.pmid: 18211953