Liu Chunyan, Chen Zhaobo, Hu Xiaoyan, Wang Lina, Li Chaoyang, Xue Jing, Zhang Pengju, Chen Weiwen, Jiang Anli
Department of Biochemistry and Molecular Biology, Medical School of Shandong University, Jinan, Shandong 250012, P.R. China.
Mol Med Rep. 2015 Jun;11(6):4625-32. doi: 10.3892/mmr.2015.3332. Epub 2015 Feb 11.
The present study aimed to investigate whether microRNA (miR)‑185 downregulated androgen receptor expression in the LNCaP prostate carcinoma cell line. Human prostate cancer (PCa) LNCaP cells were cultured and transfected with synthetic has‑miR‑185 mimic or inhibitor. The transfected cells were subsequently evaluated with a viability assay, nuclear staining, reverse transcription quantitative polymerase chain reaction (RT‑qPCR), dual luciferase assay and western blot analysis. The results of the western blot analysis and RT‑qPCR indicated that transfection with an miR‑185 mimic markedly reduced the androgen receptor (AR) protein expression levels in LNCaP cells, whereas transfection with an miR‑185 inhibitor increased the protein expression of AR in the LNCaP cells. The results of the luciferase reporter assay demonstrated that the predicted target site in the AR 3' untranslated regions was a specific functional binding site for miR‑185, and that AR was a direct target of miR‑185. In addition, downregulation of AR by miR‑185 impaired the interaction between AR and androgen response element, and downregulated the expression of the AR target gene prostate specific antigen. Data also suggested that the downregulation of AR mediated by miR‑185, inhibited the proliferation and induced the apoptosis of the LNCaP cells. Therefore, the results of the present study suggested that miR‑185 may be a potential negative modulator of AR‑mediated signaling and may act as a tumor suppressor in prostate cancer cells.
本研究旨在调查微小RNA（miR）-185是否下调LNCaP前列腺癌细胞系中的雄激素受体表达。培养人前列腺癌（PCa）LNCaP细胞，并用合成的has-miR-185模拟物或抑制剂进行转染。随后，通过活力测定、核染色、逆转录定量聚合酶链反应（RT-qPCR）、双荧光素酶测定和蛋白质印迹分析对转染后的细胞进行评估。蛋白质印迹分析和RT-qPCR结果表明，用miR-185模拟物转染可显著降低LNCaP细胞中雄激素受体（AR）蛋白表达水平，而用miR-185抑制剂转染则可增加LNCaP细胞中AR的蛋白表达。荧光素酶报告基因测定结果表明，AR 3'非翻译区的预测靶位点是miR-185的特异性功能结合位点，且AR是miR-185的直接靶标。此外，miR-185介导的AR下调削弱了AR与雄激素反应元件之间的相互作用，并下调了AR靶基因前列腺特异性抗原的表达。数据还表明，miR-185介导的AR下调抑制了LNCaP细胞的增殖并诱导其凋亡。因此，本研究结果表明，miR-185可能是AR介导信号传导的潜在负调节因子，并可能在前列腺癌细胞中作为肿瘤抑制因子发挥作用。
