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G. Li, P. Liton, C. Luna, D. L. Epstein, P. Gonzalez; Biological Effects of miR-183 in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):474.
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We have previously proposed that cellular senescence induced by oxidative stress may contribute to some of the functional alterations in the trabecular meshwork (TM) in aging and glaucoma. Profiling of microRNA (miRNAs) expression changes during stress-induced senescence after treatment with H2O2 revealed significant up-regulation of several miRNAs including miR-183. Here, we investigated whether up-regulation of this miRNA could contribute to alterations in the phenotype of HTM cells.
Primary HTM cells were transfected with either miR-183 mimic or a scrambled control using nucleofector transfection technique (Amaxa). Gene microarray analysis was conducted in triplicate using Affymetrix HG-133A2.0 arrays. The down-regulation of genes identified in the array analysis that were also present in any of the MIRANDA, TARGETSCAN, and PICTAR-VERT were validated by real-time PCR. Confirmation of the targeting of miR-183 to the 3’UTR of the selected genes was done using the psiCHECK2 dual-luciferase assay. Protein expression was measured by Western blot. Cell proliferation was evaluated using a BrdU ELISA assay kit. Phagocytic activity was measured by internalization and acidification of the pHrodo E.coli BioParticles.
Gene microarray analysis of TM cells transfected with miR-183 showed down-regualtion of several predicted targets for this miRNA including: Intergrin beta-1 (ITGB1), polo-like kinase 1 (PLK1), Kinesin heavy chain member 2A (KIF2A) and presenilin 2 (Alzheimer disease 4) (PSEN2). Targeting of the 3’ UTR regions of these four genes by miR-183 was confirmed by psiCHECK2 dual-luciferase assay. Decreased expression of ITGB1 and KIF2A in HTM cells tranfected with miR-183 was further confirmed by Western blot. HTM cells transfected with miR-183 showed distinct features that include an extensive increase in cytoplasmic vacuolization observed by light and EM microscopy, a significant decrease in proliferation, and increased phagocytic activity.
The observed effects of miR-183 in HTM cells suggest that the changes in expression of this, and potentially other miRNAs, associated with the induction of cellular senescence may play an important role in shaping the phenotypic alterations of senescent HTM cells, which, in turn, may contribute to pathophysiological changes of the TM in aging and glaucoma.
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