BACKGROUND BRD7 is a tumor suppressor known to inhibit cell proliferation and cell cycle progression and initiate apoptosis in breast cancer. However, the function and underlying molecular events of BRD7 in tumor invasion and metastasis in breast cancer are not fully understood. METHODS BRD7 expression was assessed in two stable cell lines MDA231 and MCF7 with BRD7 overexpression and one stable cell line MDA231 with BRD7 interference using qRT-PCR and western blotting. CCK8 assay was used to examine the proliferation ability of MDA231 and MCF7 cells. Scratch wound healing assay was used to evaluate cell migration in MDA231 and MCF7 cells. Both Matrigel and three-dimensional invasion assays were performed to investigate the cell invasion ability after BRD7 overexpression or silencing or YB1 restoration in MDA231 and MCF7 cells. The potential interacting proteins of BRD7 were screened using co-immunoprecipitation combined with mass spectrometry and verified by co-immunoprecipitation in HEK293T cells. Additionally, we confirmed the specific binding region between BRD7 and YB1 in HEK293T cells by constructing a series of deletion mutants of BRD7 and YB1 respectively. Finally, xenograft and metastatic mouse models using MDA231 cells were established to confirm the effect of BRD7 on tumor growth and metastasis. RESULTS Here, the results of a series of assays in vitro indicated that BRD7 has the ability to inhibit the mobility, migration and invasion of breast cancer cells. In addition, YB1 was identified as a novel interacting protein of BRD7, and BRD7 was found to associate with the C-terminus of YB1 via its N-terminus. BRD7 decreases the expression of YB1 through negatively regulating YB1 phosphorylation at Ser102, thereby promoting its proteasomal degradation. Furthermore, gene set enrichment analysis revealed that epithelial-mesenchymal transition (EMT) is the common change occurring with altered expression of either BRD7 or YB1 and that BRD7 represses mesenchymal genes and activates epithelial genes. Moreover, restoring the expression of YB1 antagonized the inhibitory effect of BRD7 on tumorigenicity, EMT, invasiveness and metastasis through a series of in vitro and in vivo experiments. Additionally, BRD7 expression was negatively correlated with the level of YB1 in breast cancer patients. The combination of low BRD7 and high YB1 expression was significantly associated with poor prognosis, distant metastasis and advanced TNM stage. CONCLUSIONS Collectively, these findings uncover that BRD7 blocks tumor growth, migration and metastasis by negatively regulating YB1-induced EMT, providing new insights into the mechanism by which BRD7 contributes to the progression and metastasis of breast cancer.

中文翻译：

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BRD7通过负调控YB1诱导的上皮-间质转化抑制乳腺癌的侵袭和转移。

背景技术BRD7是已知的抑制乳腺癌细胞增殖和细胞周期进程并启动细胞凋亡的肿瘤抑制剂。然而，BRD7在乳腺癌的肿瘤侵袭和转移中的功能和潜在的分子事件尚不完全清楚。方法采用qRT-PCR和western blotting方法，在两种稳定表达BRD7的稳定细胞系MDA231和MCF7和一种表达BRD7干扰的稳定细胞系MDA231中评估BRD7的表达。采用CCK8法检测MDA231和MCF7细胞的增殖能力。使用刮擦伤口愈合测定法评估MDA231和MCF7细胞中的细胞迁移。进行了Matrigel和三维侵袭试验，以研究BRD7过表达或沉默或YB1恢复后MDA231和MCF7细胞的细胞侵袭能力。使用免疫共沉淀结合质谱法筛选BRD7的潜在相互作用蛋白，并通过免疫共沉淀在HEK293T细胞中进行验证。另外，我们通过分别构建一系列的BRD7和YB1缺失突变体，确定了HEK293T细胞中BRD7和YB1之间的特异性结合区。最后，建立了使用MDA231细胞的异种移植和转移小鼠模型，以证实BRD7对肿瘤生长和转移的影响。结果在此，一系列体外测定的结果表明，BRD7具有抑制乳腺癌细胞的迁移，迁移和侵袭的能力。此外，YB1被鉴定为BRD7的新型相互作用蛋白，并且发现BRD7通过其N末端与YB1的C末端缔合。BRD7通过负调控Ser102处的YB1磷酸化来降低YB1的表达，从而促进其蛋白酶体降解。此外，基因集富集分析表明，上皮-间质转化（EMT）是BRD7或YB1表达改变引起的常见变化，并且BRD7抑制间充质基因并激活上皮基因。此外，通过一系列体外和体内实验，恢复YB1的表达可拮抗BRD7对致瘤性，EMT，侵袭性和转移的抑制作用。此外，乳腺癌患者中BRD7表达与YB1水平呈负相关。低BRD7和高YB1表达的组合与不良预后，远处转移和晚期TNM分期显着相关。结论集体而言，