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R. Kutty, W. Samuel, C. Jaworski, T. Duncan, C. N. Nagineni, B. Wiggert, T. M. Redmond; MicroRNA Expression in Human Retinal Pigment Epithelial Cells: Increased Expression of MicroRNA-9 (mir-9) During N-(4-Hydroxyphenyl)retinamide-Induced Apoptosis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4439.
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MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNA molecules exerting profound effects on gene expression by regulating the stability and/or the translation of target mRNAs. While expression profiles for protein-coding genes have been well documented, the information currently available regarding miRNA expression in eye tissues is limited. In order to extend our knowledge of the RPE transcriptome, we generated a miRNA profile of human RPE cell line, ARPE-19. This provided a framework for investigating possible roles for miRNAs in RPE cell apoptosis. We examined changes in miRNA expression after treatment with the synthetic retinoid N-(4-hydoxyphenyl)retinamide (4HPR).
Total RNA was isolated from cultured ARPE-19 cells with TRIzol Reagent. Small RNA enriched using the PureLink miRNA Isolation Kit was analyzed by microarray hybridization employing NCode miRNA Labeling System and NCode Multi-Species miRNA Microarrays (Invitrogen). The cells were treated with 0-20 µM 4HPR for 24 hours and the total RNA fraction was isolated using miRNeasy Kit (Qiagen). Real-time PCR analysis of miRNAs, heme oxygenase-1 (HO-1) mRNA and growth arrest and DNA damage-inducible transcription factor 153 (GADD153) mRNA were performed using TaqMan reagents (Applied Biosystems).
Microarray analysis revealed 62 miRNAs expressed at substantial levels in ARPE-19 cells, 12 of which were among the miRNAs reported to be neural retina specific by Xu et al (J. Biol. Chem. 282: 25053, 2007). Validation of expression of selected miRNAs was carried out by real-time PCR analysis. Treatment of the RPE cells with 4HPR resulted in apoptosis characterized by a marked increase in the expression of HO-1 and GADD153 transcripts. The expression of selected miRNAs during 4HPR-induced apoptosis was analyzed by real-time PCR. Interestingly, a 2-fold increase in the expression of mir-9 was observed, and the response was dependent on 4HPR concentration. However, other miRNAs tested failed to show noticeable changes in expression in response to 4HPR treatment.
Our results demonstrate that a large number of miRNAs are expressed in RPE cells in culture and that the mir-9 expression is regulated during the 4HPR-induced apoptosis. Thus, miRNAs could play an important role in RPE pathophysiology.
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