Objective: To observe the effects of Casitas B lymphoma (CBL) protein on proliferation, migration and invasion of breast cancer cells and explore its mechanism of action.
Methods: Cultured breast cancer cell lines MDA-MB-231 and MCF7A were transfected with a CBL-overexpressing plasmid and a specific siRNA targeting CBL (siRNA-CBL), respectively, and the changes in cell proliferation, migration and invasion were examined using colony-forming assay, cell counting kit-8 (CCK-8), scratch test and Transwell assay. Flow cytometry and Western blotting were performed to examine the effects of CBL overexpression on cell cycle and epithelial-mesenchymal transition (EMT) of MDA-MB-231 cells, and the changes in the number of filamentous pseudopodia were observed by rhodamine- labeled phalloidin staining of the cytoskeleton. IP-mass spectrometry identified NCK2 as the interacting proteins of CBL, and their interaction was verified by immunoprecipitation and immunofluorescence co-localization experiments in HEK-293T cells transfected with the plasmids for overexpression of CBL, NCK2, or both. Cycloheximide tracking and ubiquitination assays were used for assessing the effects of CBL on stability and ubiquitination of NCK2 protein in MDA-MB-231 cells; CCK-8 and Transwell assays were used to determine the effect of NCK2 overexpression on CBL-mediated proliferation and migration of the cells.
Results: The proliferation, migration and invasion were significantly suppressed in MDA-MB-231 cells overexpressing CBL (P < 0.05) and significantly enhanced in MCF7 cells with CBL silencing (P < 0.01). Silencing of CBL promoted G1/S transition in MCF7 cells (P < 0.05). Overexpression of CBL significantly decreased the expressions of CDK2/4 (P < 0.01), cyclinA2/B1/D1/D3/E2 (P < 0.05), Snail, N-cadherin, claudin-1 (P < 0.05), and upregulated the expression of E-cadherin (P < 0.05). CBL silencing upregulated the expressions of CDK2/4/6 (P < 0.05), cyclin A2/B1/D1/D3/E2 (P < 0.05), Snail, vimentin, and claudin-1 (P < 0.05) and down-regulated E-cadherin expression (P < 0.05). CBL overexpression obviously reduced the number of filamentous pseudopodia in MDA-MB-231 cells, and the reverse changes were observed in MCF7 cells with CBL silencing. In MDA-MB-231 cells, CBL overexpression lowered NCK2 protein stability (P < 0.05) and promoted its ubiquitin-mediated degradation (P < 0.01). Overexpression of NCK2 obviously reversed CBL-mediated inhibition of cell proliferation and migration (P < 0.01).
Conclusion: CBL can inhibit the proliferation, migration and invasion of breast cancer cells through ubiquitination-mediated degradation of NCK2.
目的: 阐明Casitas B系淋巴瘤（CBL）蛋白对乳腺癌细胞增殖、迁移和侵袭的影响并探讨其作用机制。
方法: 乳腺癌细胞系MDA-MB-231、MCF7常规培养，实验分为NC组、过表达CBL组和siNC组、siCBL组，采用克隆形成法和细胞计数试剂盒-8（CCK-8）、伤口愈合实验、Transwell实验检测过表达（沉默）CBL对乳腺癌细胞增殖、迁移和侵袭能力的影响；流式细胞术和Western blot验证CBL对乳腺癌细胞周期进程、细胞周期标志物和上皮-间充质转化（EMT）的影响；罗丹明标记的鬼笔环肽染色细胞骨架观察CBL对细胞丝状伪足数量的影响。采用IP-质谱法筛选CBL的相互作用蛋白并确定NCK2为研究对象。实验分为CBL组、NCK2组、CBL+NCK2组，通过免疫共沉淀（IP）和免疫荧光共定位实验验证CBL与NCK2蛋白互作情况。通过环己酰亚胺追踪实验和泛素化实验检测CBL对NCK2蛋白稳定性和泛素化的影响。采用CCK-8和Transwell实验检测NCK2对CBL介导的乳腺癌细胞增殖及迁移能力的影响。
结果: 过表达CBL组的细胞增殖、迁移、侵袭能力显著降低（P < 0.05）；沉默CBL组的细胞增殖、迁移、侵袭能力显著增加（P < 0.01）。沉默CBL促进MCF7细胞周期G1/S转换（P < 0.05）。过表达CBL下调乳腺癌细胞周期相关蛋白CDK2/4（P < 0.01）、Cyclin A2/B1/D1/D3/E2（P < 0.05）、EMT相关蛋白Snail、N-Cadherin、Claudin-1（P < 0.05），同时上调E-Cadherin（P < 0.05）；沉默CBL上调乳腺癌细胞周期相关蛋白CDK2/4/6（P < 0.05）、Cyclin A2/B1/D1/D3/E2（P < 0.05），EMT相关蛋白Snail、Vimentin、Claudin-1（P < 0.05），同时下调E-Cadherin（P < 0.05）。过表达CBL使乳腺癌细胞系MDA-MB-231中丝状伪足数量减少；沉默CBL使乳腺癌细胞系MCF7中丝状伪足数量增加。NCK2与CBL相互作用。过表达CBL抑制NCK2蛋白稳定性（P < 0.05），并促进其泛素降解（P < 0.01）。过表达NCK2能够逆转CBL介导的乳腺癌细胞增殖和迁移的抑制作用（P < 0.01）。
结论: CBL可通过泛素化降解NCK2抑制乳腺癌细胞增殖、迁移和侵袭。
Keywords: CBL; E3 ligase; cell invasion; cell proliferation; tyrosine kinase non-catalytic region protein 2; ubiquitylation.