Hras12V转基因小鼠肝癌发生的性别差异性蛋白质组学研究

Differential proteomic analysis of gender-dependent hepatocellular carcinoma in Hras12V transgenic mice

目的肝细胞癌(hepatocellular carcinoma,HCC)的发生具有显著的性别差异,但其分子机制尚不清楚。本研究通过检测Hras12V转基因小鼠雌雄肝肿瘤组织的蛋白质组学表达差异,探讨肝肿瘤发生的性别差异机制。方法采用双向荧光差异凝胶电泳(two dimension difference gel electrophoresis,2D-DIGE)和基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术检测Hras12V雌雄小鼠肝肿瘤组织的蛋白质组学差异表达谱,分离并鉴定差异表达蛋白,应用蛋白质印迹法验证2D-DIGE结果,并对差异表达蛋白进行生物信息学分析,包括蛋白功能注释、分类(gene ontology,GO)分析、京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路分析。结果经双向荧光差异凝胶电泳-图像分析得到1 313个蛋白点,其中差异倍数≥1.5(P<0.05)的蛋白点553个。从中选择94个差异蛋白点进行MALDI-TOF-MS鉴定,得到56种差异性表达的蛋白质。其中,与雌鼠相比,雄鼠肝肿瘤组织中高表达的蛋白有43种,低表达的蛋白有13种。选取4个差异蛋白FABP1、Albumin、Prdx6和CALR进行了蛋白质印迹法验证,其中FABP1在雄鼠肝癌中显著上调,Albumin和Prdx6在雄鼠肝癌中显著下调,CALR在雌雄小鼠肝癌中差异无统计学意义,结果与质谱数据基本一致,证明了2D-DIGE结果的可靠性。对雌雄鼠肝肿瘤组织中的差异蛋白进行GO分析,细胞组分分析显示,差异蛋白主要分布在细胞质、细胞溶质、线粒体和细胞核中;分子功能分析显示,差异蛋白主要承担poly(A)RNA结合、ATP结合和酶结合等功能;生物过程分析显示,差异蛋白主要参与运输、脂代谢过程和凋亡过程的负调控等。KEGG通路分析结果表明,差异蛋白涉及9条信号通路,主要通路包括代谢途径、内质网加工过程和细胞色素P450的异物代谢等通路。结论 Hras12V转基因小鼠雌雄肝肿瘤组织的差异性蛋白表达的检测,为研究性别差异性肝癌发生分子机制提供了基础生物学信息和有益的线索。

OBJECTIVE An outstanding characteristic of hepatocellular carcinoma （HCC） is male prevalence. How- ever, the underlying molecular mechanisms remain largely unknown. The present study is aimed to identify the differential proteomie expression of gender-dependent HCC in Hrasl2V transgenic mice and to investigate the underlying molecular mechanisms. METHODS Two-Dimensional Fluorescence Difference Gel Electrophoresis （2D-DIGE） and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry （MALDI-TOF-MS） were used to identify the differentially expressed proteins in the liver tumor tissues of male and female Hras12V transgenic mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics,including Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG）. RESULTS Among auto-detected 1313 protein spots by 2D-DIGE,553 protein spots were differentially expressed （ratio 1. 5, P〈 0. 05） between the liver tumor tissues of male and female mice,in which 94 spots were randomly selected for MALDI-TOF-MS identification and finally 56 distinct proteins were obtained. Compared with females,43 and 14 proteins were up- or down- regulated in males respectively. Four differentially expressed proteins were selected and validated by Western blot. Among them,FABPl was significantly up regulated, Albumin and Prdx6 were significantly down-regulated,and CALR had no significantly changed in male hepatic tumors comparing with female hepatic tumors. These results basically confirmed the reliability of 2D-DIGE results. GO a nalysis showed that the differentially expressed proteins in the tumor tissues between male and female mice associated to various cellular component （cytoplasm, nucleus, mitochondrion, cytosol, et al）, molecular function [poly（A） RNA binding, ATP binding, enzyme binding, et all and biological process （transport,lipid metabolism process, negative regulation of ap- optosis process, et al）. Nine pathways （Metabolic pathways, Protein processing in endoplasmic reticulum, Metabolism of xenobiotics by cytochrome P450,Oxidative phosphorylation,et al） were significantly different between the tumor tissues of males and females depending on KEGG analysis. CONCLUSION The proteomic data and relative analysis of the hepatic tumor tissues of Hras12V transgenic male and female mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on HCC.