蛋白质组学方法分析PRL-3功能相关蛋白

目的 蛋白质组学方法分析转染肝再生磷酸酶-3(PRL-3)后结肠癌细胞LoVo的蛋白表达改变.方法 构建绿色荧光蛋白载体PAcGFP-C3-PRL-3并转染至结肠癌细胞LoVo中,获得稳定表达GFP-PRL-3的结肠癌细胞LoVo-PRL-3.Western blot检测转染后细胞的PRL-3表达.采用双向凝胶电泳(2-DE)分离LoVo-PRL-3细胞及转染空载体PAcGFP-C3的LoVo-Control细胞总蛋白,从中选取差异表达蛋白质点,通过基质辅助激光解吸电离飞行时间串联质谱(MALD I-TOF-TOF-MS)鉴定差异表达的蛋白质.结果 Western blot结果显示PRL-3在LoVo-PRL-3细胞中高表达,而未检测到在LoVo-Control细胞中表达.通过双向凝胶电泳图谱分析发现20个差异蛋白质斑点,质谱成功鉴定出17种蛋白.与LoVo-Control细胞比较,10种蛋白在LoVo-PRL-3细胞中高表达,而7种蛋白在LoVo-PRL-3细胞中表达降低.结论 成功鉴定出PRL-3功能相关蛋白,为进一步研究PRL-3促进结直肠癌转移机制奠定基础.

Comparative proteomics analysis of PRL-3 functionally related protein

Objective To identify the protein expression patterns after the colon cancer cells LoVo transfected with phosphatase of regenerating liver-3 ( PRL-3 ) using a comparative proteomic approach. Methods The green fluorescent protein vector PAcGFP-C3 -PRL-3 was constructed and transfected into the colon cancer cells LoVo and colon cancer cells stably expressing GFP-PRL-3 (LoVo-PRL-3) were established. Western blotting was used to detect the expression levels of PRL-3 in LoVo-PRL-3 cells. 2-DE was applied to separate the whole proteins in LoVo-PRL-3 cells and LoVo control cells that trasfected with vectors PAcGFP-C3. The proteins with statistically significant differential expression between LoVo-PRL-3 and LoVo control cells were identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALD I-TOF-TOF-MS). Results Western blotting showed that PRL-3 was highly expresssed in LoVo-PRL-3 cells, but undetectable in LoVo control cells. Twenty proteins spots were found as differentially expressed proteins using two-dimensional gel electrophoresis and 17 proteins were successfully identified using mass spectrometry. Among these identified proteins, 10 proteins were significantly up-regulated , while 7 proteins were notably down-regulated in the lovo-PRL-3 compared to the lovo control cells.Conclusion The PRL-3 functionally related proteins were successfully identified, which laying the foundation for further studies on PRL-3-promoted metastasis.