人肝癌细胞敲低Rab27B后的差异蛋白质组初步研究

Differentially quantitative proteome of MHCC97H after Rab27B knock-down: a preliminary study

目的通过分析敲低Rab27B后肝癌细胞蛋白质表达的变化,研究其在肿瘤细胞中的作用及可能的分子机制。方法在人肝癌细胞系MHCC97H中利用Tet-on系统和RNA干扰(RNAi)技术敲低Rab27B表达,利用i TRAQ定量蛋白质组学技术比较敲低前后肿瘤细胞蛋白表达的差异,并对其进行亚细胞定位、生物过程和分子功能等生物信息学分析。结果 MHCC97H细胞敲低Rab27B后共鉴定到448种差异蛋白[比值(|Ratio|)>1.21,P<0.05],其中表达趋势与Rab27B正相关的蛋白229种,负相关的蛋白219种。差异蛋白主要涉及囊泡运输、大分子定位、胞内应激等生物过程,有26种差异蛋白分布于8个肿瘤相关信号通路中,其中11种集中在黏着斑(focal adhesion)信号通路。结论对Rab27B敲低前后MHCC97H细胞全蛋白的i TRAQ分析表明,Rab27B不仅能影响肿瘤细胞的外泌体分泌,还能引起与细胞增殖和迁移等相关的信号通路中重要蛋白的变化,提示Rab27B确实具有调控肿瘤细胞增殖和迁移的分子基础。

Objective To discover the vital role of Rab27 B in tumor cells and its potential molecular mechanism by means of quantitative proteomics analysis of Rab27 B knockdown in MHCC97 H. Methods The expression of Rab27 B in MHCC97 H cells was knocked down by the combination of Tet-on advanced inducible expression system and RNA interference technology. Then,proteins extracted from the cells were identified by LC-MS / MS system after FASP digestion and i TRAQ 4-plex labeling. Finally, the properties of differentially expressed proteins, including the subcellular localizations,biological processes and molecular functions,were analyzed by the bioinformatics method. Results There were 448 differentially expressed proteins（ ｜ Ratio ｜ 1. 21,P 0. 05） identified in MHCC97 H cells after Rab27 B knockdown. The expression levels of 229 or 219 proteins were positively or negatively correlated with Rab27 B,respectively.These differentially expressed proteins were mainly involved in vesicle transport,macromolecule localization,cellular response to stimulus. Furthermore,there were 26 differentially expressed proteins participating in 8 tumor-related signal pathways,eleven of which were in the focal adhesion signal pathway. Conclusion The analysis of quantitative proteomics in Rab27B-knockdown MHCC97 H cell line by i TRAQ suggests that Rab27 B not only has an impact on the exosomal secretion of tumor cells,but also regulates master proteins in signal pathways involved in cell proliferation and migration.