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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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Expression of miR-9 increases in ARPE-19 cells following treatment with 5-aza-2’-deoxycytidine. Cells were treated with 1 μM 5-aza-2’-deoxycytidine for 3 days, and miR-9 expression was estimated using real time RT–PCR. The expression of TGM2, a gene known to be upregulated by this treatment, was also analyzed. A: 5-aza-2’-deoxycytidine increased miR-9 expression. B: 5-aza-2’-deoxycytidine increased TGM2 expression. *p<0.05 compared to control, n=4.
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f5: Expression of miR-9 increases in ARPE-19 cells following treatment with 5-aza-2’-deoxycytidine. Cells were treated with 1 μM 5-aza-2’-deoxycytidine for 3 days, and miR-9 expression was estimated using real time RT–PCR. The expression of TGM2, a gene known to be upregulated by this treatment, was also analyzed. A: 5-aza-2’-deoxycytidine increased miR-9 expression. B: 5-aza-2’-deoxycytidine increased TGM2 expression. *p<0.05 compared to control, n=4.

Mentions: The expression of miR-9 genes could be suppressed by the hypermethylation of their promoter regions [10]. Treatment of cells with 5-aza-2’-deoxycytidine, a methyl transferase inhibitor, can alleviate this suppression. The expression of miR-9 in ARPE-19 cells increased approximately threefold in response to the treatment (Figure 5). TGM2, a gene known to be regulated by hypermethylation [28], was also induced as expected.


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)

Expression of miR-9 increases in ARPE-19 cells following treatment with 5-aza-2’-deoxycytidine. Cells were treated with 1 μM 5-aza-2’-deoxycytidine for 3 days, and miR-9 expression was estimated using real time RT–PCR. The expression of TGM2, a gene known to be upregulated by this treatment, was also analyzed. A: 5-aza-2’-deoxycytidine increased miR-9 expression. B: 5-aza-2’-deoxycytidine increased TGM2 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

f5: Expression of miR-9 increases in ARPE-19 cells following treatment with 5-aza-2’-deoxycytidine. Cells were treated with 1 μM 5-aza-2’-deoxycytidine for 3 days, and miR-9 expression was estimated using real time RT–PCR. The expression of TGM2, a gene known to be upregulated by this treatment, was also analyzed. A: 5-aza-2’-deoxycytidine increased miR-9 expression. B: 5-aza-2’-deoxycytidine increased TGM2 expression. *p<0.05 compared to control, n=4.
Mentions: The expression of miR-9 genes could be suppressed by the hypermethylation of their promoter regions [10]. Treatment of cells with 5-aza-2’-deoxycytidine, a methyl transferase inhibitor, can alleviate this suppression. The expression of miR-9 in ARPE-19 cells increased approximately threefold in response to the treatment (Figure 5). TGM2, a gene known to be regulated by hypermethylation [28], was also induced as expected.

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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