免疫沉淀——质谱联用揭示RCC2在乳腺癌MDA-MB-468细胞系中的新功能

Immunoprecipitation-mass Spectrometry Reveals New Functions of RCC2 in Breast Cancer Cell Line MDA-MB-468

目的分析RCC2蛋白在乳腺癌MDA-MB-468细胞系中的互作蛋白组并揭示其功能调控网络。方法运用酶切技术结合一代测序构建携带Flag标签RCC2蛋白过表达载体,慢病毒包装感染构建Flag-RCC2过表达的MDA-MB-468稳转细胞株。采用Flag抗体凝胶珠亲和沉淀,收集蛋白样品进行高敏感度质谱鉴定。生物信息蛋白组学分析互作蛋白组,包括GO注释,KEGG通路富集,构建蛋白互作网络(PPIN)。结果高可信度164个蛋白聚类分析显示,RCC2及其互作蛋白显著富于核糖体相关通路中,具有RNA与核蛋白体结合能力,揭示其参与蛋白翻译与成熟的过程。结论成功鉴定RCC2互作蛋白组,揭示RCC2在乳腺癌细胞中发挥新的翻译调控功能。

Objective To analyze the protein interactome and reveal the functional regulation network of RCC2 protein in MDA-MB-468 cell line. Methods Using enzyme digestion combined with one generation sequencing, we successfully constructed overexpression vector of RCC2 protein with a Flag tag. MDAMB- 468 cell line with Flag-RCC2 stably overexpression was constructed using a lentiviral vector system. Flag-tag gel beads affinity precipitation was used to collect RCC2-interacting proteins for high-sensitivity mass spectrometry analysis. And bioinformatic analyses of interacting-proteins, including GO annotation, KEGG pathway enrichment, construction of protein-protein interaction network, were further processed. Results Clustering analyses of 164 proteins with high confidence indicated that RCC2 and its interacting proteins were significantly enriched in ribosomal-related pathways and had the abilities to bind with RNA and ribosomal proteins, revealing its involvement in protein translation and maturation. Conclusion We systematically identified the RCC2 interactome map and revealed that RCC2 plays a new role of translational regulation in breast cancer cells.