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Lorenz Latta, Karl Nordström, Tanja Stachon, Fries Fabian, nora Szentmáry, Berthold Seitz, Barbara Käsmann-Kellner; A siRNA primary based cell model to confirm results from mRNA sequencing of Aniridia limbal epithelial cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2263.
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© ARVO (1962-2015); The Authors (2016-present)
PAX6-related Aniridia is a sight-threatening disease due to progression of secondary glaucoma and aniridia related keratopathy (ARK). Changes or loss of limbal epithelial progenitors causes the epithelial surfaces defects. We analyzed how PAX6 contribute to this with a two-step approach: 1) mRNA sequencing of limbal epithelial cells isolated from controls and aniridia patients. 2) confirming if the identified potential regulated RNAs are changed in siRNA based primary aniridia cell model as well. With this approach long-term and secondary effects (inflammation, vascularization changes in keratocytes) on transcription level should be distinguishable from direct PAX6 influence on limbal epithelial cell, revealing potential gene targets controlled by PAX6 leading to keratopathy in case of PAX6 deficiency.
Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium. Normal cells were obtained from limbus region of cadaveric controls. For the siRNA based cell model, cells were transfected with siRNA against PAX6 or scrambled control. All cells were lysed to obtain RNA and Protein. Reduction of PAX6 protein was controlled by Western Blot. Aniridia and control RNA-libraries were subjected to Next Generation Sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparing to NCBI GEO Datasets, annotation with DAVID and literature search. For the resulting filtered Gene list qPCR primer were ordered and postulated changes were verified with qPCR on siRNA based aniridia cell model.
We could identify gene groups which might be regulated by PAX6. Exemplary we could show that SPINK7 a protease inhibitor is downregulated in aniridia patients as well as in our anirida cell model. DSG1 was reduced in patient and model cells as expected from literature. DKK1, PITX1 and KRT12 showed different regulation in patients compared to the siRNA cell model.
The cell model is a tool to identify PAX6 targets which have a direct effect on ARK pathogenesis. This provides evidence that not all changes encountered in patients are due to substitution with conjunctival progenitor cells. Further studies are necessary to explore the influence of PAX6 on ARK in epithelial cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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