Introduction: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by Finasteride for patients with BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter region contains a CpG island, we hypothesized that somatic methylation of the promoter region may account for absence of SRD5A2 expression and DNA methyltransferases may play a role in silencing SRD5A2 expression.
Methods: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. Expression of SRD5A2 and DNA methyltransferases were analyzed by Western blot and quantitative RT-PCR. Methylation of SRD5A2 promoter region was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cells (BPH-1). To determine the effect of CpG methylation, SDR5A2 promoter-luciferase contrsucts were methylated in vitro using M.SssI methylase. IL-6 expression was analyzed using ELISA.
Results: Benign prostatic tissues from wild-type mice of different ages (n=5 per group) were harvested. We found that expression of SRD5A2 and DNMT 1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, 40% of mice at age of 12 months (2/5) demonstrated low SRD5A2 expression and high DNMT1 expression. Expression of DNMT3a and 3b were not altered. Consistently, the methylation status of SRD5A2 promoter was confirmed by Methylated CpG Island Recovery Assay (MIRA). To study the role of DNMT1 in silencing of SRD5A2, we silenced DNMT1 using siRNA in BPH-1 cells. Silencing DNMT1 led to re-expression of SRD5A2. Similarly, exposure of cells to 5-AZA-C led to re-expression of SDR5A2. The 5-AZA-C only led to increased expression of DNMT1 and not not DNMT3a or DNMT3b in BPH-1 cells. We used SDR5A2 promoter-luciferase contrsucts that were methylated in vitro using M.SssI methylase technique. When the methylated SDR5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SDR5A2 promoter caused reduction of luciferase gene expression by 80%, suggesting that methylation of the SDR5A2 promoter regulates the expression of SDR5A2. Since age-associated increased IL-6 has been shown to regulate DNMT-1, we next evaluated expression of IL-6 in aging prostate tissue. We found that expression of IL-6 was positively correlated with expression DNMT-1 in benign prostatic tissues.
Conclusions: Expression of SRD5A2 in aging prostates is dynamic. Absence of SRD5A2 expression is associated with methylation of the gene's promoter region, which is regulated by DNMT1 and not DNMT3a or DNMT3b. Dynamic and absence of SRD5A2 has implications for management of BPH and chemopreventive strategies for prostate cancer.
Citation Format: Rongbin Ge, Zongwei Wang, Chin-Lee Wu, Shahin Tabatabaei, Aria F. Olumi. Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2976. doi:10.1158/1538-7445.AM2013-2976