p90核糖体蛋白S6激酶4及其变异体相互作用蛋白质在乳腺癌中的功能研究

Protein is the main substance of the activities of life. To achieve its function, protein can not be separated from the interaction with other proteins. Alternative splicing is an important mechanism for regulating gene expression and generating protein diversity. Our pervious study suggested that the p90 ribosomal protein S6 kinase 4 (RSK4) may be a key molecule to suppress breast cancer invasion and metastasis. RSK4 pre-mRNAs undergo alternative splicing at different sites and these splices generate different mRNA variants. Each of the RSK4 variants has its own function. Howere, the mechanism of RSK4 in breast cancer is not clear yet. We proposed that different protein complexes formed by RSK4 and its variants with other proteins interaction might play an important role in breast cancer occurrence and development. To test this hypothesis, the study will establish the technique of tandem affinity purification in mammals. Afterwards, the study will identify the RSK4-interacting and RSK4 variants-interacting proteins by mass spectral analysis, and identify the key protein by bioinformatics analysis. In addition, the study will investigate in vivo and vitro the alterations of differentiation, invasion and metastasis by modifying the expression levels of RSK4, RSK4 variants and its interaction-proteins through transfection tests on breast cancer cell lines. Through the above studies, we expect to establish new ideas and ways for further clarifying mechanism of occurrence and development of breast cancer, finding new therapeutic targets of beast caner.

蛋白质是生命活动的主要物质，蛋白质功能的实现，离不开与其它蛋白质之间的相互作用。可变剪接是调节基因表达和产生蛋白质多样性的重要机制。本课题组前期的研究提示p90核糖体蛋白S6激酶4（RSK4）可能是抑制乳腺癌侵袭转移的关键分子。RSK4前体mRNA在可变剪接作用下形成变异体，并具有不同的生物学功能，但RSK4在乳腺癌中作用机制尚未明确。我们提出RSK4及其变异体与其它蛋白通过相互作用形成不同的蛋白质复合物后在乳腺癌发生发展中发挥重要的作用。为检验这一假设，本研究拟建立哺乳动物体系中的串联亲和纯化技术，并联合质谱分析鉴定RSK4及其变异体相互作用蛋白质，经生物信息学方法分析找寻RSK4相互作用蛋白中可能的关键蛋白。通过转染技术改变乳腺癌细胞株中RSK4及其相互作用蛋白的表达，从体内外观察细胞分化、侵袭转移能力的变化。从而为进一步阐明乳腺癌发生发展机制及寻找乳腺癌治疗新靶点开辟新思路和新途径。

RSK4（p90核糖体S6蛋白激酶4）作为抑癌基因，能够抑制细胞增殖、迁移并诱导细胞的凋亡。然而，来源RSK4的剪接变异体RSK4m，在某些方面却发挥着相反的作用。由于缺乏对RSK4或RSK4m的作用机制的了解，因此对与它们产生相互作用的作用因子的研究有助于揭示它们的特别功能和作用。本研究主要利用串联亲和纯化技术获得RSK4和RSK4m的相互作用蛋白复合物，通过质谱鉴定出各具体相互作用蛋白，采用生物信息学分析的这些蛋白的相互作用关系。研究过程中，我们获得分离和鉴定出82个RSK4的互作蛋白，137个RSK4m的互作蛋白，生物信息学方法Gene Ontology 和Ingenuity Pathway Analysis分析了这些相互作用蛋白的功能以及蛋白与蛋白相互作用网络，阐明了RSK4或RSK4m与它们各自的互作蛋白参与介导多种生物学进程，尤其是细胞凋亡过程。此外，我们发现，通过比较两者的互作蛋白，相比RSK4，RSK4m参与更多的生物功能。体内体外实验对上述结果也进行了验证和证实。