Division of Structural Biology, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Mumbai, India.
Division of Structural Biology, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Mumbai, India.
J Steroid Biochem Mol Biol. 2018 Jul;181:20-27. doi: 10.1016/j.jsbmb.2018.02.012. Epub 2018 Feb 22.
Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.
富含半胱氨酸的分泌蛋白 3（CRISP3）是前列腺癌中上调最明显的基因之一。雄激素受体（AR）不仅在前列腺癌发展的初始阶段发挥重要作用，而且在去势抵抗性前列腺癌（CRPC）的晚期也发挥作用。AR 调节 CRISP3 表达的作用尚不清楚。为了了解 CRISP3 表达的调控，我们将 CRISP3 启动子的各种重叠片段克隆到 pGL3 荧光素酶报告载体中。所有构建体均在 PC3（CRISP3 阴性）和 LNCaP（CRISP3 阳性）细胞系中瞬时和稳定转染，并通过荧光素酶测定测量启动子活性。LNCaP 稳定克隆的启动子活性明显高于 PC3 稳定克隆。此外，在 CRISP3 阴性的 PC3 和 RWPE-1 细胞中，CRISP3 启动子被组蛋白去乙酰化沉默。用 DHT 处理 LNCaP 细胞导致 CRISP3 转录本和蛋白水平增加。在 LNCaP 稳定克隆中，还通过荧光素酶测定评估了 CRISP3 启动子对 AR 的依赖性。为了提供 CRISP3 启动子的表观遗传调控及其对 DHT 反应的分子证据，在 PC3 和 LNCaP 细胞中进行了 ChIP PCR。我们的结果表明，前列腺癌细胞中 CRISP3 的表达依赖于雄激素，并且在 AR 阳性细胞中，CRISP3 启动子受 AR 的表观遗传调控。
