Genetica mirna

Molecular Vision 2010; 16:1475-1486 Received 29 December 2009 | Accepted 30 July 2010 | Published 4 August 2010

© 2010 Molecular Vision

MicroRNA expression in human retinal pigment epithelial (ARPE-19) cells: Increased expression of microRNA-9 by N-(4Hydroxyphenyl)retinamide
R. Krishnan Kutty,1 William Samuel,1 Cynthia Jaworski,1 Todd Duncan,1 Chandrasekharam N. Nagineni,2 NaliniRaghavachari,3 Barbara Wiggert,1 T. Michael Redmond1
1Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD; 2Laboratory

of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD; 3Genomics Core Facility, Pulmonary and Vascular Medicine Branch, National Heart Lung and Blood Institute, National Institutes ofHealth, Bethesda, MD Purpose: MicroRNAs (miRNAs) are important regulators of many cellular functions due to their ability to target mRNAs for degradation or translational inhibition. Previous studies have reported that the expression of microRNA-9 (miR-9) is regulated by retinoic acid and reactive oxygen species (ROS). We have previously shown that N-(4-hydoxyphenyl)retinamide (4HPR), a retinoic acidderivative, induces ROS generation and apoptosis in cultured human retinal pigment epithelial (RPE) cells, known as ARPE-19 cells. The aim of the present study was to investigate the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment, and to identify other miRNAs normally expressed in these cells. Methods: ARPE-19 cells in culture were treated with 4HPR, the total RNA fractions wereisolated, and the expression of various miRNAs and mRNAs was analyzed using real-time PCR. The miRNA expression profile of ARPE-19 cells was analyzed using microarray hybridization. Results: Treatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes. A twofold increase in the expression of miR-9 was also observed during thisresponse. Potential binding sites for the transcription factors encoded by CEBPA and CEBPB genes were found to be present in the putative promoter regions of all three genes encoding miR-9. 4HPR-induced miR-9 expression was associated with parallel increases in the expression of these transcription factor genes. 5-Aza-2’-deoxycytidine, a methyl transferase inhibitor, also increased the expression ofmiR-9 in ARPE-19 cells. Microarray hybridization analysis identified let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d as the most abundant miRNAs normally expressed in ARPE-19 cells. These miRNAs are known to regulate cell growth, differentiation or development. The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, althoughthese increases were modest when compared to the increase in the expression of miR-9. Conclusions: Our studies demonstrate that miR-9 is expressed in the RPE cell line ARPE-19, and its expression is increased by a retinoic acid derivative and by an inhibitor of promoter hypermethylation. Several miRNAs with inherent ability to regulate cell growth, differentiation and development are also normallyexpressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.

MicroRNA (miRNA) is a class of single-stranded noncoding small (~22 nucleotides) RNA molecules known to regulate gene expression posttranscriptionally [1,2]. The miRNAs, encoded by genes localized to various chromosomes, are initially transcribed as primary transcripts (pri-miRNAs), thenconverted to pre-miRNAs and subsequently processed to mature miRNAs, which are essential components of the RNA-initiated silencing complex (RISC). An miRNA can function as a posttranscriptional silencer of gene expression either by destabilizing its target transcripts or by inhibiting their translation. A perfect
Correspondence to: R. Krishnan Kutty, Laboratory of Retinal Cell and Molecular...