Abstract
Purpose: :
Age-related cataract is associated with oxidative stress-induced differential expression of genes. MicroRNAs (miRNAs) play a key role in modulation of gene expression at post translational level by mRNA degradation and translational inhibition. The altered expression of specific miRNAs is critical to cell differentiation as well as the development of a variety of human diseases including cataract. In the present study, we examined the expression level of miRNAs and influence of their expression level in gene expression in lens epithelial cells isolated cataractous and non-cataractous lenses from Shumiya cataract rat (SCR), and have explored the effect of oxidative stress on miRNA expression.
Methods: :
All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. LECs were isolated from cataractous or non cataractous lenses of SCR rats of identical age. Real-time PCR was used to analyze the expression levels of individual miRNA species expression level in cataractous and non cataractous LECs as well as LECs exposed to H2O2 for variable time. Inhibition of each specific miRNA (miR29a, 29c, 126, 551b, let7b, let7c) was performed using miRIDIAN Hairpin inhibitor. Protein expression of each target protein was analyzed with Western analysis using their specific antibodies.
Results: :
Real-time PCR analysis revealed the differential expression profiles of miRNA in LECs from cataractous lenses and non-cataractous ones. Expression of several miRNAs, miR29a, miR 29c, miR126 are decreased in cataractous LECs. H2O2-stressed LECs displayed increased expression of miR29 at 24h, while the expression level of miR29 decreased after 48h. Inhibition of miR29a and 29c induced the expression of tropomyosin 1α protein, a cytoskeletal protein and one of target proteins for miR29a and 29c, suggesting oxidative stress is involved in regulating tropomyosin1α expression.
Conclusions: :
Taken together, our results suggest that miRNAs expression pattern might play a major regulatory role in cataractogenesis of rat lens, and oxidative stress alters miRNA expression and thereby alters gene expression in rat LECs.
Keywords: cataract • oxidation/oxidative or free radical damage • differentiation