应用蛋白质组学技术筛选鉴定锌指蛋白139调控的胃癌转移相关蛋白质

Screening and identification of proteins related to gastric cancer metastasis with comparative proteomics

目的以胃癌SGC7901细胞原位移裸鼠模型为对象,通过荧光双向差异凝胶电泳(2D-DIGE)结合液质联用(LC-MS)质谱分析技术鉴定ZNF139调控的胃癌转移相关蛋白质。方法合成针对ZNF139的小干扰RNA(ZNF139-si RNA),以ZNF139-si RNA转染人胃癌细胞株SGC7901,G418筛选。以ZNF139-si RNA质粒转染的胃癌细胞、阴性质粒转染的胃癌细胞及普通胃癌细胞分别进行裸鼠胃癌原位移植。造模成功后取出原位移植瘤及腹腔转移淋巴结。荧光双向差异凝胶电泳(2D-DIGE)技术分别分离ZNF139-si RNA各组原位移植瘤及腹腔转移淋巴结蛋白质;选定差异点,胶内酶解后,液质联用(LC-MS)质谱分析技术鉴定蛋白质。蛋白印迹(Western blot)技术验证差异蛋白质的表达。结果转染ZNF139-si RNA质粒后SGC7901细胞中ZNF139的表达受到有效抑制。与阴性质粒组及空白组比较,阳性质粒组原位移植瘤生长更慢(P<0.05),且腹腔淋巴结转移率更低(P<0.05)。蛋白质组学结果发现在阳性质粒组原发灶中Fascin、hn RNPA2/B1表达下调,ANXA1表达上调;阳性质粒组转移淋巴结中ANXA5表达下调(P<0.05)。Western blot验证结果与蛋白质组学结果相符。结论 ZNF139可能通过调节Fascin、hn RNPA2/B1、ANXA1、ANXA5促进胃癌淋巴结转移。

Objective To screen and identify the proteins related with tumor metastasis of gastric cancer in a nude mouse model bearing orthotopic transplanted tumor. Methods Zinc finger protein 139（ZNF139）- specific si RNA was synthesized and transfected into gastric cancer cell line SGC7901, which was then screened by G418. ZNF139-si RNA-transfected cells, negative plasmid- transfected cells and untreated SGC7901 cells were orthotopically transplanted separately on the stomach wall of BALB/c nude mice. The primary tumors and metastatic lymph nodes were harvested to separate the proteins by 2- D fluorescence difference gel electrophoresis（2- D DIGE）; after gel digestion, the differential proteins were subjected to liquid chromatography- mass spectrometry（LC- MS） for identification and their functions were analyzed. Western blotting was performed to verify the identified proteins. Results ZNF139 expression was effectively inhibited in si RNA- transfected SGC7901 cells. ZNF139- si RNA- transfected cells showed obviously suppressed tumor growth with a lowered lymph node metastasis rate in nude mice compared with untreated cells and the negative control cells（P〈0.05）. Proteomic study with 2-D DIGE showed that fascin and hn RNPA2/B1 were down-regulated while ANXA1 was up-regulated in the primary tumors, and ANXA5 was down-regulated in the metastatic lymph nodes in ZNF139-si RNA-transfected group. Western blotting confirmed the results of proteomic analysis. Conclusion ZNF139 gene may promote lymph node metastasis of gastric cancer by regulating fascin, hn RNPA2/B1, ANXA1, and ANXA5.