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Impressum
After vitreous treatment cultured human RPE cells downregulate the expression of ten genes
Kociok N., Hueber A., Esser P., Thumann G., Welsandt G., Kirchhof B.
Dept. of Vitreoretinal Surgery, University of Cologne, Germany.
Purpose: Exposure of RPE cells to the vitreous is an important
risk factor for proliferative vitreoretinopathy. In order to identify
genes in human RPE cells with altered expression due to treatment with
human vitreous we carried out a differential mRNA expression analysis
(DEmRNA-PCR).
Method: Cultured human passage 3 RPE cells were incubated with
25% human vitreous for 48 h or with serum-free medium as a control. Total
RNA was prepared from these cells followed by DEmRNA-PCR, polyacrylamide
gel electrophoresis, and silver staining of the resulted bands. The differentially
amplified cDNAs were identified by DNA sequencing and gene bank search.
The gene expression of these genes in RPE cells in vivo was verified by
RT-PCR and the level of mRNA expression was quantified by Real Time RT-PCR,
normalized to GAPDH expression.
Results: The mRNA expression of 8 of the 10 genes in RPE cells
in vivo was shown here for the first time by RT-PCR. The reduced mRNA
expression due to vitreous treatment of the nuclear factor I-B2 (NFIB2),
the ankyrin-repeat containing protein KE03, the phosphatidylinositol glycan
of complementation class B (PIG-B), the DKFZp564B1462, the leukotriene-A4
hydrolase (LKHA4), the microtubule-associated protein 1B (MAP1B), the
RAS-GTPase activating protein (GAP)-binding protein (G3BP), the protein
associated with Myc (PAM), the inactive variant of the E2 ubiquitinconjugating
enzymes (UEV-1) varied from 0,69 to 0,17 compared to the expression of
the corresponding genes in untreated cells. The UDPGalNAc: polypeptide
N-acetylgalactosaminyltransferase T1 (UDP-GalNAc- T1) mRNA showed no differential
expression in treated and untreated cells.
Conclusions: DEmRNA-PCR in combination with Real Time-RT-PCR is
an effective method for detecting and verifying differentially expressed
genes without the need for preconceived assumptions. The altered expression
of the identified genes in vitreous-treated RPE cells may be a part of
a process that finally ends in PVR. Elucidating the functions of these
genes in RPE cells might open new strategies for their therapy.
Supported by DFG (Es 82/5-3, He 840/6-3), Köln Fortune program and
Retinovit Foundation