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Haijiang Lin, Bernard F. Godley; Microrna-335 Inhibits Sod2 Expression And Increases Oxidant-induced Rpe Cell Injury. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6474.
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Increased production of reactive oxygen species (ROS) over time may be associated with early pathogenesis of RPE cells in age-related macular degeneration (AMD). The development of early AMD in manganese superoxide dismutase (SOD2) knockdown mouse has provided more direct evidence of this relationship. MicroRNAs (miRNAs) negatively regulate a wide variety of genes through post-transcription inhibition. The purpose of this study is to investigate the role of miR-335 in regulating SOD2 expression and modulating RPE cell survival in response to oxidative damage.
Expression of miR-335 levels in macular RPE cells from normal young, normal old and AMD patient donors were analyzed by qRTPCR. Cultured human ARPE-19 cells were transfected with miR-335 mimic or inhibitor. Cell viability was assessed by the MTT assay. Apoptosis was determined by incubating cells with hydrogen peroxide (H2O2). DNA fragmentation was measured using an enzyme linked immunosorbent assay. SOD2 protein level was analyzed using Western blot.
MiR-335 expression was remarkably up-regulated (P<0.05) in macular RPE cells from old donors compared to RPE cell from young donors. The macular RPE cells from AMD patients exhibited a higher expression level than that of macular RPE cells from normal age-matched donors. H2O2-induced ARPE-19 cell death and apoptosis were increased by miR-335 mimic and decreased by miR-335 inhibitor. Computational analysis found a putative target site of miR-335 in the 3’UTR of SOD2 mRNA, which was verified by luciferase reporter assay. Forced over-expression of miR-335 decreased SOD2 expression level and increased H2O2 cell death. This effect was blocked by abrogation of miR-335.
The findings highlight that over-expression of miR335 inhibits the expression of SOD2 and may play an important role in the development of AMD. Therefore, miR335 may serve as a potential target for pharmaceutical intervention in AMD.
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