Vitreous treatment of cultured human RPE cells results in differential expression of 10 new genes.
OBJECTIVE
To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous.


METHODS
Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR.


RESULTS
Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells.


CONCLUSIONS
Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein-protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.
Kociok
Norbert
N
Department of Vitreoretinal Surgery, Division of Ophthalmology, University of Cologne, Germany. norbert.kociok@uni-koeln.de
Hueber
Arno
A
Esser
Peter
P
Schraermeyer
Ulrich
U
Thumann
Gabriele
G
Luther
Thomas T
TT
Jordan
Jens
J
Welsandt
Gerhard
G
Kirchhof
Bernd
B
D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014822:Vitreous Body / Q000502:physiology*
D017931:DNA Primers; D005136:Eye Proteins; D012333:RNA, Messenger