Discovering genes and pathways involved in maintenance of epithelial phenotype in retinal pigment epithelium cells

Authors

• JR Chang
• BS Jha
• M Lal
• R Sharma
• M Ferrer
• K Bharti

Abstract

Proliferative vitreoretinopathy (PVR) is a blinding disease in which retinal pigment epithelium (RPE) de-differentiates and loses its epithelial characteristics. The goal of this study is to perform a series of high-throughput screening (HTS) assays and bioinformatics analyses on immature RPE to identify genes and pathways that are involved in promoting epithelial phenotype. For HTS, human induced-pluripotent stem cells (iPSCs)-derived RPE expressing GFP tagged to RPE-specific gene and RFP tagged to a constitutive housekeeping gene was optimized for culture in 384-well plates. A library of 875 miRNA mimics was tested, and top 5% GFP up- and down-regulators were identified. Multiple miRNA databases were used to analyze these selected mimics for their target genes. Targets that had >90% scores and common occurrences between multiple databases per miRNA were chosen for subsequent screenings using siRNA. siRNA screening results and Ingenuity Pathway Analysis (IPA) software were used to compile a list of genes for small molecule assays. In our results, miRNA assays showed a high reproducibility with correlation coefficients of 0.87, 0.94, 0.91 in GFP, RFP, Hoechst signals, respectively, between two identical screenings. miRNA database analyses and IPA results predicted genes and pathways that are involved in regulating the RPE epithelial phenotype. siRNA screening comprising of 122 siRNAs further confirmed these findings and emphasized genes involved in epithelial-to-mesenchymal transition (EMT) and tight junctions. Small molecule inhibitors and activators of the selected genes validated the results and discovered a unique set of genes that modulate epithelial phenotype of human RPE.

Scientific Focus Area: Molecular Biology and Biochemistry

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