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Nigar Fatma, Eri Kubo, Bhavana Chhunchha, Biju Bhargavan, Dhirendra P. Singh; Prdx6-Deficiency in Lens Epithelial Cells Causes Oxidative stress-Related Altered Expression of MicroRNAs: Role of MiRNA22. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1041.
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Peroxiredoxin (Prdx) 6 provides cytoprotection by optimizing reactive oxygen species (ROS)-mediated etiopathology. MicroRNAs (miRs) affect diverse biological processes. The effects of Prdx6 deficiency or elevated ROS expression on miRs expression and the role of their targeted mRNA in lens epithelial cells (LECs) are unclear. Using Prdx6-depleted cells as a model for redox-active (aging) cells and LECs facing oxidative stress, we examined expression levels of miRs and their effect on gene expression in context to aging/cataractous LECs.
Prdx6+/+ and Prdx6-/- LECs were used for Multiplexed miRNA profiling assay (Asuragen Inc., Austin, TX). Results were validated by Real-time PCR. Target Scan Human 5.2 and Genecopoeia database identified target genes. Changes in expression of miRs and target genes were analyzed by Real-time PCR and Western analysis. Transfection assays with miRs overexpression or inhibitors defined functionality. Transcriptional activity of miRs target genes was measured by CAT-ELISA. MTS assay was used for cell viability. Apoptosis was measured by FITC Annexin-V Apoptosis Detection Kit. H2DCFH-DA dye was used to measure ROS levels.
Prdx6-/- cells displayed higher levels of ROS and were under oxidative stress. miRNA array analysis showed 22 and 10 miRs either up or downregulated (>2 fold), respectively in Prdx6-/- cells compared to Prdx6+/+ and >8 miRs showed 4- to 5-fold increase or decrease. The significantly upregulated miRs (miR22, miR27b, miR200abc, miR412, miR466d-3P, miR759 and miR669h-5P) were related to oxidative stress-induced pathologies and apoptotic cell death. miR133a, miR496 and miR495 were downregulated and related to development and aging. Real-time PCR using total RNA from lenses of human subjects aged 16 and 75 showed a similar pattern of expression to Prdx6+/+ vs Prdx6-/- LECs. Overexpression of miR22 in Prdx6+/+ cells caused reduced expression of Prdx6 with increased levels of ROS and TGFβRI and increased expression of TGFβs inducible gene, βig-h3 and αSMA, markers of cataractogenesis. These processes were attenuated by exogenous Prdx6 or its inducer, curcumin.
Results suggest that Prdx6-deficiency causes oxidative stress-related aberrant expression of miRs, which erroneously regulates gene expression in Prdx6-/- LECs or in aging LECs. miRs may play a role in cataractogenesis related to ROS or Prdx6-deficiency.
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