BACKGROUND We have previously shown that the zinc finger transcription repressor SNAI2 (SLUG) represses tumor suppressor BRCA2-expression in non-dividing cells by binding to the E2-box upstream of the transcription start site. However, it is unclear how proliferating breast cancer (BC) cells that has higher oxidation state, overcome this repression. In this study, we provide insight into the mechanism of de-silencing of BRCA2 gene expression by PRDX5A, which is the longest member of the peroxiredoxin5 family, in proliferating breast cancer cells. METHODS We used cell synchronization and DNA affinity pulldown to analyze PRDX5A binding to the BRCA2 silencer. We used oxidative stress and microRNA (miRNA) treatments to study nuclear localization of PRDX5A and its impact on BRCA2-expression. We validated our findings using mutational, reporter assay, and immunofluorescence analyses. RESULTS Under oxidative stress, proliferating BC cells express PRDX5 isoform A (PRDX5A). In the nucleus, PRDX5A binds to the BRCA2 silencer near the E2-box, displacing SLUG and enhancing BRCA2-expression. Nuclear PRDX5A is translated from the second AUG codon in frame to the first AUG codon in the PRDX5A transcript that retains all exons. Mutation of the first AUG increases nuclear localization of PRDX5A in MDA-MB-231 cells, but mutation of the second AUG decreases it. Increased mitronic hsa-miRNA-6855-3p levels under oxidative stress renders translation from the second AUG preferable. Mutational analysis using reporter assay uncovered a miR-6855-3p binding site between the first and second AUG codon in the PRDX5A transcript. miR-6855-3p mimic increases accumulation of nuclear PRDX5A and inhibits reporter gene translation. CONCLUSION Oxidative stress increases miR-6855-3p expression and binding to the inter-AUG sequence of the PRDX5A transcript, promoting translation of nuclear PRDX5A. Nuclear PRDX5A relieves SLUG-mediated BRCA2 silencing, resulting in increased BRCA2-expression.

中文翻译：

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氧化还原/miR-6855-3p/PRDX5A 轴在逆转乳腺癌细胞中 SLUG 介导的 BRCA2 沉默中的作用。

背景我们之前已经表明，锌指转录抑制因子 SNAI2 (SLUG) 通过与转录起始位点上游的 E2 盒结合来抑制肿瘤抑制因子 BRCA2 在非分裂细胞中的表达。然而，尚不清楚增殖的具有较高氧化态的乳腺癌 (BC) 细胞如何克服这种抑制。在这项研究中，我们深入了解了 PRDX5A（过氧化物酶 5 家族中最长的成员）在增殖的乳腺癌细胞中对 BRCA2 基因表达的去沉默机制。方法 我们使用细胞同步和 DNA 亲和力下拉来分析 PRDX5A 与 BRCA2 消音器的结合。我们使用氧化应激和 microRNA (miRNA) 处理来研究 PRDX5A 的核定位及其对 BRCA2 表达的影响。我们使用突变、报告分析验证了我们的发现，和免疫荧光分析。结果 在氧化应激下，增殖的 BC 细胞表达 PRDX5 亚型 A (PRDX5A)。在细胞核中，PRDX5A 与 E2 盒附近的 BRCA2 消音器结合，取代 SLUG 并增强 BRCA2 表达。核 PRDX5A 从框内的第二个 AUG 密码子翻译成保留所有外显子的 PRDX5A 转录物中的第一个 AUG 密码子。第一个 AUG 的突变增加了 MDA-MB-231 细胞中 PRDX5A 的核定位，但第二个 AUG 的突变降低了它。在氧化应激下增加的 mitronic hsa-miRNA-6855-3p 水平使得从第二个 AUG 开始的翻译更可取。使用报告基因测定的突变分析发现了 PRDX5A 转录本中第一个和第二个 AUG 密码子之间的 miR-6855-3p 结合位点。miR-6855-3p mimic 增加核 PRDX5A 的积累并抑制报告基因翻译。结论 氧化应激增加 miR-6855-3p 表达并结合 PRDX5A 转录物的 AUG 间序列，促进核 PRDX5A 的翻译。核 PRDX5A 解除 SLUG 介导的 BRCA2 沉默，导致 BRCA2 表达增加。