Howell Jennifer C, Chun Eugene, Farrell Annie N, Hur Elizabeth Y, Caroti Courtney M, Iuvone P Michael, Haque Rashidul
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Mol Vis. 2013;19:544-60. Epub 2013 Mar 15.
In recent years, microRNAs (miRNAs) have been reported to play important roles in a broad range of biologic processes, including oxidative stress-mediated ocular diseases. In addition, the polyphenolic compound curcumin has been shown to possess anti-inflammatory, antioxidant, anticancer, antiproliferative, and proapoptotic activities. The aim of this study was to investigate the impact of curcumin on the expression profiles of miRNAs in ARPE-19 cells exposed to oxidative stress.
MiRNA expression profiles were measured in ARPE-19 cells treated with 20 μΜ curcumin and 200 μΜ H₂O₂. PCR array analysis was performed using web-based software from SABiosciences. The cytotoxicity of ARPE-19 cells was determined with the CellTiter-Blue cell viability assay. The effects of curcumin on potential miRNA targets were analyzed with quantitative real-time PCR and western blotting.
Curcumin treatment alone for 6 h had no effect on ARPE-19 cell viability. Incubation with H₂O₂ (200 µM) alone for 18 h decreased cell viability by 12.5%. Curcumin alone downregulated 20 miRNAs and upregulated nine miRNAs compared with controls. H₂O₂ downregulated 18 miRNAs and upregulated 29 miRNAs. Furthermore, curcumin pretreatment in cells exposed to H₂O₂ significantly reduced the H₂O₂-induced expression of 17 miRNAs. As determined with quantitative real-time PCR and western blotting, curcumin increased the expression of antioxidant genes and reduced angiotensin II type 1 receptor, nuclear factor-kappa B, and vascular endothelial growth factor expression at the messenger RNA and protein levels.
The results demonstrated that curcumin alters the expression of H₂O₂-modulated miRNAs that are putative regulators of antioxidant defense and renin-angiotensin systems, which have been reported to be linked to ocular diseases.
近年来，据报道微小RNA（miRNA）在广泛的生物学过程中发挥重要作用，包括氧化应激介导的眼部疾病。此外，多酚化合物姜黄素已被证明具有抗炎、抗氧化、抗癌、抗增殖和促凋亡活性。本研究的目的是探讨姜黄素对暴露于氧化应激的ARPE - 19细胞中miRNA表达谱的影响。
在经20μM姜黄素和200μM过氧化氢处理的ARPE - 19细胞中测量miRNA表达谱。使用SABiosciences基于网络的软件进行PCR阵列分析。用CellTiter - Blue细胞活力测定法测定ARPE - 19细胞的细胞毒性。用定量实时PCR和蛋白质印迹分析姜黄素对潜在miRNA靶标的影响。
单独用姜黄素处理6小时对ARPE - 19细胞活力无影响。单独用200μM过氧化氢孵育18小时使细胞活力降低12.5%。与对照组相比，单独使用姜黄素下调了20种miRNA并上调了9种miRNA。过氧化氢下调了18种miRNA并上调了29种miRNA。此外，在暴露于过氧化氢的细胞中进行姜黄素预处理可显著降低过氧化氢诱导的17种miRNA的表达。通过定量实时PCR和蛋白质印迹测定，姜黄素在信使RNA和蛋白质水平上增加了抗氧化基因的表达，并降低了血管紧张素II 1型受体、核因子κB和血管内皮生长因子的表达。
结果表明，姜黄素改变了过氧化氢调节的miRNA的表达，这些miRNA是抗氧化防御和肾素 - 血管紧张素系统的假定调节因子，据报道它们与眼部疾病有关。
