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Guorong Li, Coralia Luna, Jing Wu, Jianming Qiu, David L. Epstein, Pedro Gonzalez; Anti-inflammatory Role of miR-493 in Senescent Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1428.
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We hypothesize that cellular senescence involved pathogenically in the outflow pathway in glaucoma. MicroRNA (miRNA) miR-493 is consistently up-regulated during replicative senescence in human trabecular meshwork (HTM) cells. However, there is currently no information about its potential biological function. The objective of this study was to gain insight on the cellular functions regulated by miR-493.
Levels of miRNA expression in replicative senescent HTM cells were evaluated by TaqMan Q-PCR. Targeting of the 3’UTR of IL8 by miR-493 was studied by dual-luciferase reporter assay. Changes in gene expression induced by miR-493 were analyzed using gene arrays and validated by Q-PCR. Protein levels of IL6, IL8 and IL11 were evaluated by ELISA.
Senescent HTM cells (passage 15) showed an increase in expression of miR-493 (24.91±6.82 fold in HTM636 and 3.4±0.14 fold in HTM1073, both p<0.05) compared to young HTM cells (passage 4). Dual-luciferase assay indicated that IL8 is a direct target of miR-493. Gene array analysis showed that miR-493 significantly down-regulated multiple inflammatory mediators such as IL1beta, IL1RN, IL6, IL8, IL11, IL12A, IL33, CXCL1, 2, 3, 5 and 6 as well as IGF1, IGFBP and MYOC. Down-regulation of IL6, IL8, IL11 and MYOC was confirmed by real-time Q-PCR in three independent HTM cell lines (IL6: 2.91±0.57, 2.42±0.52, 2.12±0.14 fold; IL8: 5.13±1.28, 5.42±2.29, 6.38±0.59 fold; IL11: 1.66±0.66, 1.61±0.23, 3.26±0.23 fold; and MYOC: 3.5±0.84, 2.78±0.52, 2.69±0.44 fold, respectively). MiR-493 significantly decreased protein levels of IL6, IL8 and IL11 in the cell culture media (IL6: 6.55±0.57, 1.99±0.47, 1.35±0.2 folds; IL8: 4.01±0.31, 2.15±0.63, 2.82±0.19 fold; IL11: 2.48±0.51, 1.5±0.31, 2.44±0.27 fold, respectively).
Our results suggest that miR-493 acts as an anti-inflammatory miRNA in HTM cells. The observed up-regulation of miR-493 in senescent HTM cells could represent a homeostatic mechanism aimed at preventing an excessive expression of inflammatory mediators induced by the chronic activation of a stress response characteristic of the TM in glaucoma donors.
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