目的:含溴结构域蛋白7(BRD7)在包括乳腺癌在内的许多类型的癌症中下调并作为肿瘤抑制因子发挥作用,BRD7表达异常与乳腺癌的发生发展密切相关。尽管很少关注乳腺癌中BRD7蛋白水平的调节,这需要进一步阐明。
方法:用蛋白质印迹法检测乳腺癌细胞中BRD7蛋白的稳定性和乳腺癌组织中BRD7蛋白的水平。在MDA-MB-231细胞中通过共免疫沉淀结合质谱分析筛选与BRD7相互作用的潜在E3泛素连接酶蛋白。我们通过免疫共沉淀(Co-IP)和免疫荧光测定证明了BRD7与含28的三方基序(TRIM28)之间的相互作用。Co-IP和泛素化分析用于探索BRD7和TRIM28之间的特异性结合域以及BRD7的泛素化位点。通过qPCR研究TRIM28对BRD7蛋白稳定性和泛素化水平的影响,Western印迹和Co-IP测定。进行CCK-8和克隆形成测定以评估TRIM28对乳腺癌细胞增殖能力的影响。使用Transwell试验和伤口愈合试验来研究TRIM28对乳腺癌细胞侵袭和迁移的影响。流式细胞术检测TRIM28对乳腺癌细胞周期和凋亡的影响。此外,我们通过异种移植和转移小鼠模型证实了TRIM28对肿瘤生长和转移的影响。我们设计了一些恢复试验,以探讨恢复BRD7在TRIM28介导的体内和体外促进乳腺癌恶性进展中的作用。最后,免疫组化证实TRIM28和BRD7的临床意义。
结果:在这项研究中,我们证明BRD7是一种不稳定的蛋白,在乳腺癌中可能受到泛素化的调节;我们发现TRIM28的卷曲区可以直接与BRD7的N端结合,并且TRIM28通过作为潜在的E3泛素连接酶介导BRD7泛素化和依赖于K21的降解。此外,TRIM28促进细胞增殖,迁移,入侵,异种移植肿瘤生长和转移,因此在乳腺癌中起致癌作用。此外,乳腺癌中BRD7表达的恢复显著逆转了TRIM28对体内外恶性进展的促进作用.此外,TRIM28在乳腺癌活检组织中高表达,其表达与BRD7表达呈负相关,与BC患者TNM分期及不良预后呈正相关。
结论:我们的发现提供了一种新机制,通过该机制TRIM28显著促进BRD7泛素化和降解,从而促进乳腺癌恶性进展。靶向TRIM28/BRD7轴可能是乳腺癌临床诊断和治疗的一种新的潜在策略。
OBJECTIVE: Bromodomain-containing protein 7 (BRD7) is downregulated and functions as a tumor suppressor in many types of cancers including breast cancer, and the dysregulation of BRD7 expression is closely related to the development and progression of breast cancer. Whereas little attention has been focused on the regulation of BRD7 protein levels in breast cancer, which needs to be further elucidated.
METHODS: The protein stability of BRD7 in breast cancer cells and BRD7 protein level in breast cancer tissues was examined by Western Blotting. The potential E3 ubiquitin ligase proteins that interact with the BRD7 was screened by coimmunoprecipitation combined with mass spectrometry analysis in MDA-MB-231 cells. We proved the interaction between BRD7 and tripartite motif containing 28 (TRIM28) through Co-Immunoprecipitation (Co-IP) and immunofluorescence assays. Co-IP and ubiquitination assay were used to explore the specific binding domain between BRD7 and
TRIM28 and the ubiquitination site of BRD7. The effects of
TRIM28 on the BRD7 protein stability and ubiquitination level was investigated by qPCR, Western Blot and Co-IP assay. CCK-8 and clone formation assays were carried out to assess the effect of TRIM28 on proliferation ability of breast cancer ells. Transwell assay and wound healing assay were used to investigate the effect of TRIM28 on breast cancer cell invasion and migration. Flow cytometry was used to detect the effect of TRIM28 on cell cycle and apoptosis of breast cancer cells. In addition, we confirmed effect of
TRIM28 on tumor growth and metastasis by xenograft and metastatic mouse models. We designed some recovery assays to explore the role of recovery BRD7 in
TRIM28-mediated promotion of malignant progression of breast cancer in vivo and in vitro. Finally, the clinical significance of
TRIM28 and BRD7 was proved by immunohistochemistry.
RESULTS: In this study, we demonstrated that BRD7 was an unstable protein and might be regulated by ubiquitination in breast cancer; furthermore, we found that the Coiled-Coil region of TRIM28 could directly bind to N-terminal of BRD7, and TRIM28 mediates BRD7 ubiquitination and degradation dependent on K21 by acting as a potential E3 ubiquitin ligase. Moreover, TRIM28 promoted cell proliferation, migration, invasion, xenograft tumor growth and metastasis, thus playing an oncogenic role in breast cancer. Furthermore, the restoration of BRD7 expression in breast cancer significantly reversed the promotional effects of TRIM28 on malignant progression both in vitro and in vivo. In addition, TRIM28 was highly expressed in the biopsy tissues of breast cancer, and its expression was negatively correlated with BRD7 expression and positively correlated with TNM stage and poor prognosis of BC patients.
CONCLUSIONS: Our findings provide a novel mechanism by which TRIM28 significantly facilitates BRD7 ubiquitination and degradation, thus promoting breast cancer malignant progression. Targeting the TRIM28/BRD7 axis might be a novel potential strategy for the clinical diagnosis and treatment of breast cancer.