Robo1 与Nek9相互作用并影响胃癌细胞迁移

目的:筛选与Robo1分子相互作用的蛋白质,为揭示Robo1调节胃癌细胞迁移能力的机制提供线索。方法:通过Transwell迁移实验检测下调Robo1对胃癌细胞SGC7901迁移能力的影响,并对胃癌细胞SGC7901利用Robo1抗体和lgG分别进行免疫沉淀,再经质谱检测及生物信息学分析筛选与Robo1分子特异性结合的蛋白质,通过免疫共沉淀(Co-immunoprecipitation,Co-IP)验证蛋白质相互作用。结果:(1)转染Robo1小干扰RNA(si RNA)的SGC7901细胞较转染阴性对照RNA的迁移能力显著降低。(2)通过免疫沉淀、质谱检测和生物信息学分析的两次生物学重复得到一系列与Robo1相互作用的蛋白,筛选出丰度高、重复性好且功能相关的Nek9蛋白。(3)Co-IP实验结果显示与对照抗体相比,Robo1抗体可特异性地将Nek9沉淀下来,同时Nek9抗体也可特异性地将Robo1共沉淀下来,确证了Robo1-Nek9的相互作用。结论:Nek9是与Robo1相互作用的蛋白质,Robo1下调影响人胃癌细胞系SGC7901的迁移能力,可能通过Nek9起作用。Robo1-Nek9的相互作用为进一步研究Robo1在胃癌细胞中的功能及机制提供了有利的线索。

Interaction of Robo1 with Nek9 and Their Effects on Migration of Gastric Cancer Cells

Objective:To investigate the role of Robo1 in Gastric cancer (GC) cell migration and to identify the interactive protein of Robo1, thus providing clues for better understanding of its function and mechanism.Methods:Transwell migration assay was used to examine the motility of GC cell SGC7901 after Robo1 was down-regulated. Anti-Robo1 antibody and lgG were used to conduct the immunoprecipitation assay of SGC7901 cells. Then Mass Spectrum(MS) and bioinformatics were used to screen the proteins specifically binding to Robo1. Putative interactions were further confirmed by Co-IP experiments.Results:(1) The migration of SGC79901 cells which were transfected with Robo1 siRNA was markedly inhibited compared to that were transfected with the negative control. (2) A series of proteins were identified as interactive proteins of Robo1 through two times of biologically repeated IP, MS and bioinformatics analysis. And Nek9 was chosen for further verification, for its high intensity, repeatability and related biological function. (3) The CO-IP experiments showed that anti-Robo1 antibody specifically co-immunoprecipitated Nek9 and anti-Nek9 antibody also specifically co-immunoprecipitated Robo1, with lgG as a negative control, which further confirmed the Robo1-Nek9 interaction.Conclusion:Robo1 physically interacts with Nek9 and influences the migration of SGC7901 cells, probably through a mechanism involving Nek9. Robo1-Nek9 interaction provides clues to further explore the function of Robo1 and its mechanism.