Department of Microbiology, Immunology, and Physiology, Meharry Medical College, Nashville, TN, 37208, USA.
School of Graduate Studies and Research, Meharry Medical College, Nashville, TN, 37208, USA.
Cell Commun Signal. 2020 Jan 27;18(1):15. doi: 10.1186/s12964-019-0493-5.
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.
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.
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.
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.
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 盒结合来抑制非分裂细胞中的肿瘤抑制因子 BRCA2 的表达。然而，尚不清楚具有更高氧化状态的增殖性乳腺癌（BC）细胞如何克服这种抑制。在这项研究中，我们深入研究了 PRDX5A （peroxiredoxin5 家族中最长的成员）使 BRCA2 基因表达去沉默的机制，该机制在增殖性乳腺癌细胞中起作用。
我们使用细胞同步化和 DNA 亲和拉下分析来分析 PRDX5A 与 BRCA2 沉默子的结合。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、报告基因分析和免疫荧光分析验证了我们的发现。
在氧化应激下，增殖性 BC 细胞表达 PRDX5 同工型 A（PRDX5A）。在核内，PRDX5A 与 E2 盒附近的 BRCA2 沉默子结合，取代 SLUG 并增强 BRCA2 的表达。PRDX5A 从 PRDX5A 转录本的第二个 AUG 密码子以框架方式翻译到第一个 AUG 密码子，保留所有外显子。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 的突变则减少了核定位。氧化应激下 mitronic hsa-miRNA-6855-3p 水平的增加使从第二个 AUG 进行翻译成为首选。使用报告基因分析的突变分析揭示了 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 模拟物增加核 PRDX5A 的积累并抑制报告基因翻译。
氧化应激增加了 miR-6855-3p 的表达并与 PRDX5A 转录本的两个 AUG 密码子之间的序列结合，促进核 PRDX5A 的翻译。核 PRDX5A 缓解了 SLUG 介导的 BRCA2 沉默，导致 BRCA2 表达增加。
