TY - JOUR
T1 - SFRP1 promoter methylation and expression in human trabecular meshwork cells
AU - Mao, Weiming
AU - Rubin, Jeffrey S.
AU - Anoruo, Nancy
AU - Wordinger, Robert J.
AU - Clark, Abbot F.
PY - 2012/4
Y1 - 2012/4
N2 - Glaucoma is a leading cause of blindness worldwide. In primary open angle glaucoma (POAG) patients, impaired trabecular meshwork (TM) function results in elevated intraocular pressure (IOP), which is the primary risk factor of developing optic neuropathy. Our previous studies showed that Wnt signaling pathway components are expressed in the human TM (HTM), and the Wnt inhibitor, . secreted frizzled- related protein 1 (SFRP1) is elevated in the glaucomatous TM (GTM). Elevated SFRP1 increased IOP in mice eyes and in perfusion cultured anterior segments of the human eye. However, the cause of elevated . SFRP1 in the GTM remains unknown. Promoter methylation plays a key role in regulating . SFRP1 expression in certain cancer cells. In light of this, we studied whether promoter methylation is also involved in . SFRP1 differential expression in the TM. Two normal TM (NTM) and two GTM cell strains were cultured for an additional 7 days after they were confluent. RNA and genomic DNA (gDNA) were isolated simultaneously to compare . SFRP1 expression levels by quantitative PCR (qPCR) and to determine . SFRP1 promoter methylation status by bisulfite conversion and methylation-sensitive high resolution melting analysis (MS-HRM). To study whether DNA methylation inhibitors affect . SFRP1 expression in TM cells, the four TM cell strains were treated with or without 2 μM 5-aza-2'-deoxycytidine (AZA-dC) for 4 days. RNA was isolated to compare . SFRP1 expression by qPCR. In addition, a human cancer cell line, NCI-H460, was used as a positive control. We found that the two GTM cell strains had significantly higher expression levels of . SFRP1 than the two NTM cell strains. However, the . SFRP1 promoter of all four TM cell strains was unmethylated. In addition, AZA-dC treatment did not affect . SFRP1 expression in any of the TM cell strains (n = 3, . p > 0.05). In contrast, the hypermethylated . SFRP1 promoter of NCI-H460 cells was partially demethylated by the same treatment. AZA-dC treatment also elevated . SFRP1 expression by approximately two fold in NCI-H460 cells (n = 3, . p < 0.01). Our data suggest that the differential expression of . SFRP1 in HTM cells is not due to differential promoter methylation.
AB - Glaucoma is a leading cause of blindness worldwide. In primary open angle glaucoma (POAG) patients, impaired trabecular meshwork (TM) function results in elevated intraocular pressure (IOP), which is the primary risk factor of developing optic neuropathy. Our previous studies showed that Wnt signaling pathway components are expressed in the human TM (HTM), and the Wnt inhibitor, . secreted frizzled- related protein 1 (SFRP1) is elevated in the glaucomatous TM (GTM). Elevated SFRP1 increased IOP in mice eyes and in perfusion cultured anterior segments of the human eye. However, the cause of elevated . SFRP1 in the GTM remains unknown. Promoter methylation plays a key role in regulating . SFRP1 expression in certain cancer cells. In light of this, we studied whether promoter methylation is also involved in . SFRP1 differential expression in the TM. Two normal TM (NTM) and two GTM cell strains were cultured for an additional 7 days after they were confluent. RNA and genomic DNA (gDNA) were isolated simultaneously to compare . SFRP1 expression levels by quantitative PCR (qPCR) and to determine . SFRP1 promoter methylation status by bisulfite conversion and methylation-sensitive high resolution melting analysis (MS-HRM). To study whether DNA methylation inhibitors affect . SFRP1 expression in TM cells, the four TM cell strains were treated with or without 2 μM 5-aza-2'-deoxycytidine (AZA-dC) for 4 days. RNA was isolated to compare . SFRP1 expression by qPCR. In addition, a human cancer cell line, NCI-H460, was used as a positive control. We found that the two GTM cell strains had significantly higher expression levels of . SFRP1 than the two NTM cell strains. However, the . SFRP1 promoter of all four TM cell strains was unmethylated. In addition, AZA-dC treatment did not affect . SFRP1 expression in any of the TM cell strains (n = 3, . p > 0.05). In contrast, the hypermethylated . SFRP1 promoter of NCI-H460 cells was partially demethylated by the same treatment. AZA-dC treatment also elevated . SFRP1 expression by approximately two fold in NCI-H460 cells (n = 3, . p < 0.01). Our data suggest that the differential expression of . SFRP1 in HTM cells is not due to differential promoter methylation.
KW - 5-aza-2'-deoxycytidine
KW - DNA methylation
KW - Glaucoma
KW - Intraocular pressure
KW - SFRP1
KW - Trabecular meshwork
UR - http://www.scopus.com/inward/record.url?scp=84859502229&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2012.01.003
DO - 10.1016/j.exer.2012.01.003
M3 - Article
C2 - 22248913
AN - SCOPUS:84859502229
SN - 0014-4835
VL - 97
SP - 130
EP - 136
JO - Experimental Eye Research
JF - Experimental Eye Research
IS - 1
ER -