ABSTRACT
PURPOSE: To study the influence of serum-free B27 supplemented culture medium on corneal epithelial cells from limbal explants. METHODS: Human limbal tissues obtained from cadaveric donor eyes were used in this study. The morphological characteristics of cultivated epithelial cells were analyzed by phase contrast microscopy. Growth kinetics, bromodeoxyuridine (BrdU) labeling cell proliferation assay, and reverse transcriptase PCR (RT-PCR) for limbus and corneal markers were studied in serum-dependent and serum-free B27 supplemented corneal epithelial culture. The signaling pathway genes were analyzed by RT(2) qPCR profiler array. RESULTS: The corneal epithelial cells morphology and mRNA expression of markers were similar in both the serum-dependent and serum-free B27 supplemented culture. The growth and proliferation of the serum-free B27 supplemented culture was significantly higher than that of the serum-dependent culture. The wnt, hedgehog, survival, NFkB, Jak-Stat, and calcium protein kinase C pathways were highly expressed in the serum-free B27 supplemented corneal epithelial culture. CONCLUSIONS: Most signaling pathway genes are upfolded by B27 supplementation in the corneal epithelial cell culture; it could be an efficient replacement for serum.
Subject(s)
Culture Media, Serum-Free/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelium, Corneal/cytology , Signal Transduction/drug effects , Aged , Aged, 80 and over , Bromodeoxyuridine/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Epithelial Cells/cytology , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Kinetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/geneticsABSTRACT
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 (Subject(s)
Antigens, Neoplasm/metabolism
, Cell Adhesion Molecules/metabolism
, Gene Expression Regulation
, Genome, Human
, Retinoblastoma/genetics
, Retinoblastoma/metabolism
, Antigens, Neoplasm/genetics
, Azacitidine/pharmacology
, Cell Adhesion Molecules/genetics
, Cell Line
, Cell Proliferation
, Down-Regulation
, Epithelial Cell Adhesion Molecule
, Gene Silencing
, Humans
, Microarray Analysis
, Mitogen-Activated Protein Kinase Kinases/metabolism
, Retinoblastoma/pathology
, Reverse Transcriptase Polymerase Chain Reaction/methods
, Tumor Suppressor Protein p53/metabolism
, Up-Regulation
