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Patricia Sanchez-Diaz, Whitney Greene, Teresa Burke, Heuy-Ching Hetty Wang; MicroRNAs as potential regulators of fibrosis in retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3431.
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© ARVO (1962-2015); The Authors (2016-present)
This study aims to assess the role of microRNAs in the phenotypic changes observed in RPE cells after penetrating ocular trauma. We hypothesized that microRNAs (miRNAs) may mediate fibrotic changes in injured RPE cells and thus be involved in proliferative vitreoretinopathy (PVR), a leading cause of vision loss in children and young adults for which there is not a successful therapy.
We used Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia) to measure the activity of the fibrosis mediator alpha smooth muscle actin (α-SMA) promoter in ARPE19 cells transfected with miRNA mimics, hairpin inhibitors, or negative controls (Dharmacon). Activation of α-SMA promoter was assessed in the transfected cells via immunofluorescence and using mouse anti-α-SMA (primary) and AlexaFluor 568 Goat anti-Mouse (secondary) antibodies (1:100 dilution; Abcam). Gene expression was quantified via qPCR using the delta delta CT method with 18S as endogenous control. Cells were treated with the α-SMA inducer TGFB2 (10 ng/mL; positive control) and transfected with the microRNA mimic, hairpin inhibitor, or negative controls. Data was collected at days 2, 3, and 6 post-transfection. At least 2 independent experiments were performed with triplicates.
Using an in vitro model of PVR, we identified 11 differentially expressed miRNAs (DEmiRs) potentially involved in RPE fibrosis. Our luciferase reporter assay showed increased α-SMA promoter activity in ARPE19 cells after transfection with 6 of the 11 miRNA mimics compared to their negative controls (up to 70% increase at day 6). Transfection with a single miRNA hairpin inhibitor reduced the expression of the α-SMA promoter in cells treated with TGFB2 by 2-fold as well as expression of α-SMA protein at days 3 and 6 via immunofluorescence. The effect of this hairpin inhibitor on α-SMA expression was confirmed at the RNA level (~50% reduction) using a TaqMan gene expression assay (Thermofisher).
Our results were consistent with a potential role for miRNAs in the phenotypic changes associated with RPE transdifferentiation into myofibroblast-like cells. Future work aims to explore these and other DEmiRs as potential markers and therapeutic targets for PVR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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