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Reduced expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1
Rongbin Ge, MD PhD, Zongwei Wang, PhD, Aleksandar Otsetov, MD, Shulin Wu, MD PhD, Chin-lee Wu, MD PhD, Shahin Tabatabaei, MD, Aria Olumi, MD.
MGH, boston, MA, USA.

BACKGROUND:
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 bladder outlet obstrution secondary to BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter would be regulated by DNA methyltransferases leading to suppression of SRD5A2.
METHODS:
Western blot and ELISA were used to evaluate protein expression. Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, 96 prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter 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 cell line,BPH-1. To determine the effect of CpG methylation, SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. Statistical analyses were performed with JMP Pro version 11 (SAS Institute Inc., Cary, NC).
RESULTS:
Benign prostatic tissues from wild-type mice of different ages (3 months, 6 months and 12 months, n=6 per group) were harvested. We found that expression of SRD5A2 and DNMT1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, two mice at age of 12 months (2/6) had low levels of SRD5A2 with concomitant increased DNMT1 protein levels, while expression of other DNMT family members, DNMT3a and 3b, were not affected. Consistent with reduced expression of SRD5A2 and increased DNMT1, the methylation of SRD5A2 promoter was increased as assayed by MIRA. Moreover, silencing of DNMT1 with siRNA led to re-expression of SRD5A2. Similarly, exposure of BPH-1 cells to 5-AZA-C, a demethylating agent, led to re-expression of SRD5A2 and reduced expression of DNMT1, but did not affect DNMT3a or 3b expression. We used SRD5A2 promoter-luciferase constructs that were methylated in vitro using M.SssI methylase. When the methylated SRD5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SRD5A2 promoter caused reduction of luciferase expression by 80%, suggesting that methylation of the SRD5A2 promoter regulates the expression of SRD5A2.
CONCLUSIONS:
We show that SRD5A2 expression is lacking in many benign human adult prostate tissues. , Methylation of SRD5A2 promoter region, which is regulated by DNMT1, accounts of absence of SRD5A2 expression in many adult human prostate tissues. Methylation and absence to SRD5A2 expression may be used as a gene signature for tailored management of patients with BPH and chemopreventive strategies for prostate cancer.


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