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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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Increased miR-9 expression in ARPE-19 cells during 4HPR-induced apoptosis and the expression of HMOX1 and GADD153. ARPE-19 cells were treated with indicated concentrations of 4HPR for 24 h, and real-time RT–PCR was employed to analyze the expression of miR-9, HMOX1 and GADD153. Formation of mono- and oligonucleosomes was estimated using ELISA as a marker for apoptosis. A: 4HPR increased miR-9 expression. B: 4HPR-induced apoptosis as indicated by the generation of mono- and oligonucleosomes. C: 4HPR-induced HMOX1 expression. D: 4HPR-induced GADD153 expression. *p<0.05 compared to control, n=4.
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f1: Increased miR-9 expression in ARPE-19 cells during 4HPR-induced apoptosis and the expression of HMOX1 and GADD153. ARPE-19 cells were treated with indicated concentrations of 4HPR for 24 h, and real-time RT–PCR was employed to analyze the expression of miR-9, HMOX1 and GADD153. Formation of mono- and oligonucleosomes was estimated using ELISA as a marker for apoptosis. A: 4HPR increased miR-9 expression. B: 4HPR-induced apoptosis as indicated by the generation of mono- and oligonucleosomes. C: 4HPR-induced HMOX1 expression. D: 4HPR-induced GADD153 expression. *p<0.05 compared to control, n=4.

Mentions: We analyzed the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment. The cells were treated with varying concentrations of 4HPR for 24 h, and the miR-9 expression was measured using real-time RT–PCR. An increase in miR-9 expression was observed with increasing concentration of 4HPR (Figure 1). A ~2 fold increase in miR-9 expression was detected when the concentration of 4HPR reached 10 µM. Dimethyl sulfoxide, the vehicle used in this study to dissolve 4HPR, had no effect on the miR-9 expression by itself. ARPE-19 cells from passages 20 to 24 did not show detectable variation in miR-9 expression or its response to 4HPR treatment (data not shown). The 4HPR treatment induced apoptosis, as expected, as indicated by the generation of mono- and oligonucleosomes in the treated cells. We also analyzed the expression of two genes, HMOX1 (a marker for oxidative stress) and GADD153, since we have previously shown that their expression is highly increased during 4HPR-induced apoptosis of ARPE-19 cells [26]. The 4HPR-dependent increase in miR-9 expression paralleled increases in the expression of HMOX1and GADD153. Thus, treatment of ARPE-19 cells with 4HPR induces expression of miR-9 along with established markers of oxidative stress and apoptosis.


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)

Increased miR-9 expression in ARPE-19 cells during 4HPR-induced apoptosis and the expression of HMOX1 and GADD153. ARPE-19 cells were treated with indicated concentrations of 4HPR for 24 h, and real-time RT–PCR was employed to analyze the expression of miR-9, HMOX1 and GADD153. Formation of mono- and oligonucleosomes was estimated using ELISA as a marker for apoptosis. A: 4HPR increased miR-9 expression. B: 4HPR-induced apoptosis as indicated by the generation of mono- and oligonucleosomes. C: 4HPR-induced HMOX1 expression. D: 4HPR-induced GADD153 expression. *p<0.05 compared to control, n=4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Increased miR-9 expression in ARPE-19 cells during 4HPR-induced apoptosis and the expression of HMOX1 and GADD153. ARPE-19 cells were treated with indicated concentrations of 4HPR for 24 h, and real-time RT–PCR was employed to analyze the expression of miR-9, HMOX1 and GADD153. Formation of mono- and oligonucleosomes was estimated using ELISA as a marker for apoptosis. A: 4HPR increased miR-9 expression. B: 4HPR-induced apoptosis as indicated by the generation of mono- and oligonucleosomes. C: 4HPR-induced HMOX1 expression. D: 4HPR-induced GADD153 expression. *p<0.05 compared to control, n=4.
Mentions: We analyzed the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment. The cells were treated with varying concentrations of 4HPR for 24 h, and the miR-9 expression was measured using real-time RT–PCR. An increase in miR-9 expression was observed with increasing concentration of 4HPR (Figure 1). A ~2 fold increase in miR-9 expression was detected when the concentration of 4HPR reached 10 µM. Dimethyl sulfoxide, the vehicle used in this study to dissolve 4HPR, had no effect on the miR-9 expression by itself. ARPE-19 cells from passages 20 to 24 did not show detectable variation in miR-9 expression or its response to 4HPR treatment (data not shown). The 4HPR treatment induced apoptosis, as expected, as indicated by the generation of mono- and oligonucleosomes in the treated cells. We also analyzed the expression of two genes, HMOX1 (a marker for oxidative stress) and GADD153, since we have previously shown that their expression is highly increased during 4HPR-induced apoptosis of ARPE-19 cells [26]. The 4HPR-dependent increase in miR-9 expression paralleled increases in the expression of HMOX1and GADD153. Thus, treatment of ARPE-19 cells with 4HPR induces expression of miR-9 along with established markers of oxidative stress and apoptosis.

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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