Background: Breast cancer is the most common malignancy, and approximately 70% of breast cancers are estrogen receptor-α (ERα) positive. The anti-estrogen tamoxifen is a highly effective and commonly used treatment for patients with ER+ breast cancer. However, 30% of breast cancer patients fail adjuvant tamoxifen therapy and most of metastatic breast cancer patients develop tamoxifen resistance. Although increasing evidence suggests that microRNA (miRNA) dysregulation influences tamoxifen sensitivity, the mechanism of the cross-talk between miRNA and ERα signaling remains unclear. miR-575 has been reported to be involved in carcinogenesis and progression, however, the role of miR-575 in breast cancer remains limited. The aim of this study was to understand the mechanism of miR-575 in breast cancer tamoxifen resistance./nMethod: RT-qPCR was employed to assess miR-575 expression in breast cancer tissues and cell lines. The association of miR-575 expression with overall survival in patients with breast cancer was evaluated with KM plotter. Additionally, the effects of miR-575 on breast cancer proliferation and tamoxifen sensitivity were investigated both in vitro and in vivo. Bioinformatic analyses and luciferase reporter assays were performed to validate CDKN1B and BRCA1 as direct targets of miR-31-5p. The ERα binding sites in the miR-575 promoter region was validated with ChIP and luciferase assays. ERα interactions with CDKN1B, cyclin D1 or BRCA1 were determined by IP analysis, and protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively./nResults: miR-575 levels were higher in ER+ breast cancer than in ER- breast cancer and patients with high miR-575 expression had a significantly poorer outcome than those with low miR-575 expression. ERα bound the miR-575 promoter to activate its transcription, and tamoxifen treatment downregulated miR-575 expression in ER+ breast cancer. Overexpression of miR-575 decreased tamoxifen sensitivity by targeting CDKN1B and BRCA1. CDKN1B and BRCA1 were both able to antagonize ERα activity by inhibiting ERα nuclear translocation and interaction with cyclin D1. Furthermore, miR-575 expression was found to be upregulated in ER+ breast cancer cell with acquired tamoxifen resistance, whereas depletion of miR-575 partially re-sensitized these cells to tamoxifen by regulation of CDKN1B./nConclusions: Our data reveal the ERα-miR-575-CDKN1B feedback loop in ER+ breast cancer, suggesting that miR-575 can be used as a prognostic biomarker in patients with ER+ breast cancer, as well as a predictor or a promising target for tamoxifen sensitivity.

中文翻译：

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ERα-miR-575-p27 反馈回路调节 ER 阳性乳腺癌中的他莫昔芬敏感性。

背景：乳腺癌是最常见的恶性肿瘤，大约 70% 的乳腺癌是雌激素受体-α（ERα）阳性。抗雌激素他莫昔芬是一种高效且常用的治疗 ER+ 乳腺癌患者的药物。然而，30% 的乳腺癌患者辅助他莫昔芬治疗失败，大多数转移性乳腺癌患者出现他莫昔芬耐药。尽管越来越多的证据表明 microRNA (miRNA) 失调会影响他莫昔芬的敏感性，但 miRNA 和 ERα 信号传导之间的串扰机制仍不清楚。据报道，miR-575 参与致癌作用和进展，然而，miR-575 在乳腺癌中的作用仍然有限。本研究的目的是了解miR-575在乳腺癌他莫昔芬耐药中的机制。/n方法： RT-qPCR 用于评估 miR-575 在乳腺癌组织和细胞系中的表达。使用 KM 绘图仪评估 miR-575 表达与乳腺癌患者总生存期的关联。此外，在体外和体内研究了 miR-575 对乳腺癌增殖和他莫昔芬敏感性的影响. 进行生物信息学分析和荧光素酶报告基因检测以验证 CDKN1B 和 BRCA1 作为 miR-31-5p 的直接靶标。miR-575 启动子区域中的 ERα 结合位点通过 ChIP 和荧光素酶测定进行了验证。ERα 与 CDKN1B、细胞周期蛋白 D1 或 BRCA1 的相互作用通过 IP 分析确定，蛋白质表达水平和定位分别通过蛋白质印迹和免疫荧光分析。/n结果：ER+乳腺癌中的miR-575水平高于ER-乳腺癌，并且miR-575高表达患者的预后明显低于miR-575低表达患者。ERα 结合 miR-575 启动子以激活其转录，他莫昔芬治疗下调 ER+ 乳腺癌中 miR-575 的表达。miR-575 的过表达通过靶向 CDKN1B 和 BRCA1 降低了他莫昔芬的敏感性。CDKN1B 和 BRCA1 都能够通过抑制 ERα 核易位和与细胞周期蛋白 D1 的相互作用来拮抗 ERα 活性。此外，发现 miR-575 表达在具有获得性他莫昔芬耐药性的 ER+ 乳腺癌细胞中上调，而 miR-575 的消耗通过调节 CDKN1B 使这些细胞对他莫昔芬部分重新敏感。/n结论： 我们的数据揭示了 ER+ 乳腺癌中的 ERα-miR-575-CDKN1B 反馈回路，表明 miR-575 可用作 ER+ 乳腺癌患者的预后生物标志物，以及他莫昔芬敏感性的预测因子或有希望的靶标。