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MicroRNA expression in human retinal pigment epithelial (ARPE-19) cells: increased expression of microRNA-9 by N-(4-hydroxyphenyl)retinamide.

Kutty RK, Samuel W, Jaworski C, Duncan T, Nagineni CN, Raghavachari N, Wiggert B, Redmond TM - Mol. Vis. (2010)

Bottom Line: Treatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes.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 of miR-9 in ARPE-19 cells.The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retinal Cell and Molecular Biology, Bldg. 6, Room 112, National Eye Institute, National Institutes of Health, 6 Center Dr., MSC 0608, Bethesda, MD 20892, USA. kuttyk@nei.nih.gov

ABSTRACT

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-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, 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 were isolated, 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 this response. 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 of miR-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, although these 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 normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.

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Related in: MedlinePlus

Microarray hybridization analysis of miRNAs normally expressed in ARPE-19 cells. The data shown is representative of hybridization analyses performed using two RNA samples isolated from untreated ARPE-19 cells. The microarrays contained duplicate spots for each miRNA probe. The average fluorescence intensities are shown; the view is filtered by name, which has multiple members selected.
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f6: Microarray hybridization analysis of miRNAs normally expressed in ARPE-19 cells. The data shown is representative of hybridization analyses performed using two RNA samples isolated from untreated ARPE-19 cells. The microarrays contained duplicate spots for each miRNA probe. The average fluorescence intensities are shown; the view is filtered by name, which has multiple members selected.

Mentions: We next used microarray hybridization analysis to obtain a broad view of the microRNA species normally expressed in ARPE-19 cells. Small RNA preparation, which was enriched from total RNA isolated from untreated ARPE-19 cells, was employed for hybridization analysis on a microarray containing duplicate spots of each miRNA probe. The microarray analysis, when repeated using a second RNA sample, yielded similar results. The microarray data are provided at GEO (GSE23107). The relative expression levels of 62 miRNA (out of 366 human miRNAs tested) expressed substantially in these cells are shown in Figure 6. The most abundant miRNAs expressed in ARPE-19 cells were let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d. The microarray analysis failed to detect the miR-9 expression in ARPE-19 cells. This could be due to the limited sensitivity of microarray analysis in comparison to real-time RT–PCR, where the target is detected after multiple cycles of amplification.


MicroRNA expression in human retinal pigment epithelial (ARPE-19) cells: increased expression of microRNA-9 by N-(4-hydroxyphenyl)retinamide.

Kutty RK, Samuel W, Jaworski C, Duncan T, Nagineni CN, Raghavachari N, Wiggert B, Redmond TM - Mol. Vis. (2010)

Microarray hybridization analysis of miRNAs normally expressed in ARPE-19 cells. The data shown is representative of hybridization analyses performed using two RNA samples isolated from untreated ARPE-19 cells. The microarrays contained duplicate spots for each miRNA probe. The average fluorescence intensities are shown; the view is filtered by name, which has multiple members selected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2925906&req=5

f6: Microarray hybridization analysis of miRNAs normally expressed in ARPE-19 cells. The data shown is representative of hybridization analyses performed using two RNA samples isolated from untreated ARPE-19 cells. The microarrays contained duplicate spots for each miRNA probe. The average fluorescence intensities are shown; the view is filtered by name, which has multiple members selected.
Mentions: We next used microarray hybridization analysis to obtain a broad view of the microRNA species normally expressed in ARPE-19 cells. Small RNA preparation, which was enriched from total RNA isolated from untreated ARPE-19 cells, was employed for hybridization analysis on a microarray containing duplicate spots of each miRNA probe. The microarray analysis, when repeated using a second RNA sample, yielded similar results. The microarray data are provided at GEO (GSE23107). The relative expression levels of 62 miRNA (out of 366 human miRNAs tested) expressed substantially in these cells are shown in Figure 6. The most abundant miRNAs expressed in ARPE-19 cells were let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d. The microarray analysis failed to detect the miR-9 expression in ARPE-19 cells. This could be due to the limited sensitivity of microarray analysis in comparison to real-time RT–PCR, where the target is detected after multiple cycles of amplification.

Bottom Line: Treatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes.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 of miR-9 in ARPE-19 cells.The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retinal Cell and Molecular Biology, Bldg. 6, Room 112, National Eye Institute, National Institutes of Health, 6 Center Dr., MSC 0608, Bethesda, MD 20892, USA. kuttyk@nei.nih.gov

ABSTRACT

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-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, 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 were isolated, 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 this response. 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 of miR-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, although these 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 normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.

Show MeSH
Related in: MedlinePlus